Process for preparing optically active trans-3-substituted glycidic acid ester

ABSTRACT

Methods for the preparation of (2S,3S)-1,5-benzothiazepine derivatives of the formula (II) and isomers thereof, and for the preparation of (2R,3R)-1,5-benzothiazepine derivatives of the formula (III) and isomers thereof,                    
     wherein ring A and ring B are independently a substituted or unsubstituted benzene ring, and R 2  is a 2-(dimethylamino)ethyl group or a group of the formula:                    
     or a pharmaceutically acceptable salt thereof.

This application is a divisional of application Ser. No. 09/032,030,filed on Feb. 27, 1998, now U.S. Pat. No. 5,998,637 the entire contentsof which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates to a process for preparing opticallyactive trans-3-substituted glycidic acid esters. More particularly, thepresent invention relates to a process for preparing optical isomers oftrans-3-(substituted or unsubstituted phenyl)glycidic acid esters whichare useful as intermediates for the synthesis of pharmaceuticalcompounds, and the use of the optical isomers.

Diltiazem hydrochloride, the chemical name of which is (2S,3S)-3-acetoxy-5-[2-(dimethylamino)ethyl]-2,3-dihydro-2-(4-methoxyphenyl)-1,5-benzothiazepin-4(5H)-one hydrochloride, is a pharmaceutical compound widely used as acalcium channel blocker for the treatment of angina pectoris, essentialhypertension and the like (Merck Index, XII Ed., page 541).

For the preparation of diltiazem, conventionally known is a processwherein racemic trans-3-(4-methoxyphenyl)glycidic acid ester is used asthe starting material and an optical resolution is carried out at alater stage in the synthesis, as disclosed in Japanese PatentPublication Kokoku No. 46-16749, No. 53-18038 and No. 61-52142.

Also, a process using a (2R,3S)-3-(4-methoxyphenyl)glycidic acid methylester obtained by optical resolution of the racemic trans-glycidic acidester is proposed for the preparation of diltiazem in apanese PatentPublication Kokai No. 60-13776.

Thus, various processes for the preparation of optically activetrans-3-(4-methoxyphenyl)glycidic acid esters have been investigatedand, for instance, processes as mentioned below are proposed:

(a) a process comprising hydrolyzing racemictrans-3-(4-methoxyphenyl)glycidic acid methyl ester to form an alkalimetal salt, forming its diastereomeric salt with an optically resolvingreagent such as (−)-α-methylbenzylamine, resolving the salt andesterifying again the obtained optically active salt (Japanese PatentPublication Kokai No. 61-145174 and No. 2-231480),

b) a process comprising conducting a Darzens reaction of a chloroaceticacid ester having an asymmetric ester residue such as (−)-menthyl group,(−)-2-phenylcyclohexyl group or (−)-8-phenylmenthyl group withp-anisaldehyde (Japanese Patent Publication Kokai No. 61-268663, No.2-17170 and No. 2-17169),

(c) a process comprising enzymatically and asymmetrically hydrolyzing(2S, 3R)-isomer in racemic trans-3-(4-methoxyphenyl)glycidic acid methylester and recovering the remaining (2R,3S)-isomer (Japanese PatentPublication Kokai No. 2-109995 and No. 3-15398 and WO 90/04643),

(d) a process for the asymmetric synthesis of (2R,3S)-3-(4-methoxyphenyl)glycidic acid methyl ester comprising subjectingtrans-4-methoxycinnamic acid methyl ester to osmium oxidation in thepresence of an asymmetric catalyst to give an optically active diol andsubjecting the diol to intramolecular ring closure to give the desiredcompound (WO 89/02428 and WO 89/10350), and

(e) a process comprising subjecting (2S, 3R)-isomer in racemictrans-3-(4-methoxy-phenyl)glycidic acid methyl ester to enzymaticasymmetric transesterification with butanol to give (2R,3S)-3-(4-methoxyphenyl)glycidic acid methyl ester (Japanese PatentPublication Kokai No. 4-228095 and No. 6-78790).

It is also known that some 1,5-benzothiazepine derivatives other thandiltiazem have excellent pharmacological activities. For instance,Japanese Patent Publication Kokai No. 60-202871 discloses thatbenzothiazepine derivatives having a reverse absolute configuration ofdiltiazem at the 2- and 3-positions have platelet aggregation inhibitoryactivity and the like.

It is also known that (2S,3R)-3-(4-methylphenyl)glycidic acid methylester which is useful in the synthesis of this derivative is prepared byenzymatic asymmetric hydrolysis of racemictrans-3-(4-methylphenyl)glycidic acid methyl ester (Japanese PatentPublication Kokai No. 3-175995).

Japanese Patent Publication Kokai No. 8-259552 discloses a process forobtaining both isomers of trans-3-(4-methoxyphenyl)glycidic acid methylester in high optical purity from racemate by enzymatically andasymmetrically transesterifying the (2S, 3R)-isomer therein withbutanol, recovering the untransesterified (2R,3S)-3-(4-methoxyphenyl)glycidic acid methyl ester and chemicallytransesterifying the transesterified product, i.e.,(2S,3R)-3-(4-methoxyphenyl)glycidic acid butyl ester to convert into thecorresponding methyl ester.

Japanese Patent Publication Kokai No. 4-217969 discloses a process forobtaining crystals of (2R, 3S)-isomer oftrans-3-(4-methoxyphenyl)glycidic acid methyl ester by dissolving anequimolar mixture of (2R,3S)-isomer and (2S,3R)-isomer and(2R,3S)-isomer in t-butyl methyl ether solvent under heating, adding acrystalline seed of (2R,3S)-isomer, and crystallizing (2R,3S)-isomer,thereby giving crystalline (2R,3S)-isomer in an amount slightly largerthan that of (2R, 3S)-isomer initially dissolved together with theequimolar mixture.

Also, Japanese Patent Publication Kokai No. 5-301864 discloses a processfor obtaining crystals of (2R, 3S)-isomer oftrans-3-(4-methoxyphenyl)glycidic acid 4-chloro-3-methylphenyl ester bythermally dissolving an equimolar mixture of (2R, 3S)-isomer and (2S,3R)-isomer, and the (2R, 3S)-isomer in tetrahydrofuran, adding acrystalline seed of (2R, 3S)-isomer, and crystallizing (2R,3S)-isomer at30° C., thereby giving crystalline (2R,3S)-isomer in an amount slightlylarger than that of (2R,3S)-isomer initially dissolved together with theequimolar mixture.

Further, Japanese Patent Publication Kokai No. 8-259552 discloses aprocess for obtaining (2R,3S)-isomer oftrans-3-(4-methoxyphenyl)glycidic acid methyl ester from an equimolarmixture of (2R, 3S)-isomer and (2S, 3R)-isomer by enzymatically andasymmetrically transesterifying the (2S, 3R)-isomer therein with butanoluntil the molar ratio of (2S, 3R)-butyl ester/(2S,3R)-methyl ester is7.8/1, and crystallizing the (2R, 3S)-isomer therefrom.

However, in this process, in order to prevent the contamination in thedesired (2R, 3S)-methyl ester due to the crystallization of theunesterified (2S, 3R)-methyl ester remaining in a small amount, thecrystallization was stopped in a stage that the(2R,3S)-3-(4-methoxyphenyl)glycidic acid methyl ester still remains inthe mother liquor in an amount larger than the untransesterified(2S,3R)-isomer.

Thus, despite the fact that (2R,3S)-3-(4-methoxyphenyl)glycidic acidmethyl ester is scarcely transesterified in this transesterificationreaction and the transesterification conversion rate of the (2S,3R)-isomer is high, the yield of (2R,3S)-3-(4-methoxyphenyl)glycidicacid methyl ester obtained in the form of crystals is not satisfactory.

It is an object of the present invention to provide a process foroptically resolving trans-3-(substituted or unsubstitutedphenyl)glycidic acid esters in a simple manner in a high yield and inhigh optical purity.

A further object of the present invention is to provide a process forcrystallizing a desired optical isomer of trans-3-(substituted orunsubstituted phenyl)glycidic acid ester from a solution containing amixture of optical isomers thereof in high purity and in a high yieldsince the desired optical isomer can be crystallized up to the extentthat the concentration of the desired optical isomer remaining in themother liquor is extremely low as compared with known processes.

A still further object of the present invention is to provide a processfor crystallizing a desired optical isomer of trans-3-(substituted orunsubstituted phenyl)glycidic acid ester in high purity from a reactionmixture of an enzymatic asymmetric transesterification of racemate up tothe extent that the concentration of the desired optical isomerremaining in the mother liquor is extremely low as compared with knownprocesses.

These and other objects of the present invention will become apparentfrom the description hereinafter.

SUMMARY OF THE INVENTION

It is found by the inventors of the present invention that if, in asolution containing a racemic trans-3-(substituted or unsubstitutedphenyl)glycidic acid ester, and an ester compound which is differentfrom one isomer of the racemic ester only in the ester residue and whichhas a higher solubility than the isomers of the trans-3-(substituted orunsubstituted phenyl)glycidic acid ester, crystallization of an isomerhaving the same absolute configuration as the ester compound ishindered.

Thus, in accordance with the present invention, there is provided aprocess for preparing an optically active isomer of trans-3-substitutedglycidic acid ester compound of the formula (I):

wherein ring A is a substituted or unsubstituted benzene ring, and R¹ isan ester residue, which comprises:

preparing a solution of one optical isomer (A) and the other opticalisomer (B) of the ester compound (I), both of which are the opticalisomers due to the asymmetric carbons at 2- and 3-positions, and anester compound (B′) which is different from the isomer (B) only in theester residue R¹,

crystallizing the optical isomer (A) from the solution up to the extentthat the optical isomer (A) is crystallized without the precipitation ofthe optical isomer (B) due to the presence of the ester compound (B′)though the optical isomer (B) would precipitate if the ester compound(B′) were not present, and

isolating the crystals of the optical isomer (A).

The solution from which the optical isomer (A) is crystallized mayfurther contain a small amount of an ester compound (A′) which isdifferent from the optical isomer (A) only in the ester residue R¹ andhas the same ester residue as the ester compound (B′).

The solution from which the optical isomer (A) is crystallized may be asolution obtained by subjecting a solution of the optical isomers (A)and (B) and an alcohol to transesterification in the presence of anenzyme having a stereoselective transesterification ability totransesterify the isomer (B) with the alcohol, thereby producing theester compound (B′).

Thus, the present invention also provides a process for preparing anoptically active isomer of trans-3-substituted glycidic acid estercompound of the formula (I):

wherein ring A is a substituted or unsubstituted benzene ring, and R¹ isan ester residue, which comprises:

subjecting a mixture of one optical isomer (A) and the other opticalisomer (B) of the ester compound (I), both of which are the opticalisomers due to the asymmetric carbons at 2- and 3-positions, totransesterification in the presence of an alcohol and an enzyme having astereoselective transesterification ability, thereby transesterifyingthe optical isomer (B) with the alcohol to produce an ester compound(B′) which is different from the isomer (B) only in the ester residue R¹until the molar ratio of ester compound (B′)/isomer (B) falls within therange of 13/7 to 7.8/1,

crystallizing the optical isomer (A) from the resulting solutioncontaining the isomer (A), the untransesterified isomer (B) and theester compound (B′), and

isolating the isomer (A) having optical purity of at least 99% in ayield of at least 75% based on the initial amount of optical isomer (A).

The other optical isomer (B) can also be obtained in high purity and ina high yield by, after isolating the isomer (A), chemicallytransesterifying the ester compound (B′) in the mother liquor to convertinto the isomer (B) and crystallizing the isomer (B) followed byisolation thereof.

According to the present invention, an optical isomer oftrans-3-substituted glycidic acid ester (I) can be crystallized andobtained in high purity from a solution of a mixture of optical isomersof the ester (I) and an ester compound which is different from one ofthe isomers only in the ester residue, up to the extent that theconcentration of the desired optical isomer in the mother liquor becomesvery low as compared with that in conventional processes.

Further, from a racemic trans-3-substituted glycidic acid ester, thedesired isomer of high purity can be obtained in a simple manner in ahigh yield by conducting enzymatic asymmetric transesterification of theracemic ester and subsequently crystallizing the desired isomer from theresulting reaction mixture.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates the condition under which an optical isomer (A) iscrystallized from a solution (a) containing the optical isomers (A) and(B) and an ester compound (B′), but the optical isomer (B) does notprecipitate due to the presence of the ester compound (B′) though theoptical isomer (B) would precipitate in the absence of the estercompound (B′) by the use of the parameters of temperature and time.

In this connection, it is assumed that the ratio of the optical isomers(A) and (B) and the ester compound (B′) in such solution (a) issufficient for the inhibition of the crystallization of the opticalisomer (B).

DETAILED DESCRIPTION

In the present invention, a solution wherein one optical isomer (A) andthe other optical isomer (B) of a trans-3-substituted glycidic acidester of the formula (I):

in which ring A is a substituted or unsubstituted benzene ring and R¹ isan ester residue, both of which are the optical isomers due to theasymmetric carbons at the 2- and 3-positions of the ester (I), and anester compound (B′) which is different from the optical isomer (B) onlyin the ester residue are dissolved in a solvent (hereinafter referred toas “solution ABB′”) is used in crystallization.

The solution of the isomers (A) and (B) and the ester compound (B′) mayfurther contain a small amount of an ester compound (A′) which isdifferent from the isomer (A) only in the ester residue R¹ and has thesame ester residue R¹ as the ester compound (B′) (hereinafter referredto as “solution AA′BB′”).

The trans-3-substituted glycidic acid esters (I), namely a mixture ofthe optical isomers (A) and (B) thereof, used in the present inventionare the compounds of the formula (I) wherein the ring A is a substitutedor unsubstituted benzene ring and R¹ is an ester residue which enablesto crystallize the trans-3-substituted glycidic acid esters (I) in asolvent for the crystallization.

Such trans-3-substituted glycidic acid esters are, for instance,compounds of the formula (I) wherein the ring A is phenyl group whichmay be substituted by

(a) a linear or branched lower alkyl group, e.g., methyl group, ethylgroup, propyl group, isopropyl group, n-butyl group, sec-butyl group,t-butyl group, n-hexyl group, 2-hexyl group or 3-hexyl group;

(b) a linear or branched lower alkoxy group, e.g., methoxy group, ethoxygroup, propyloxy group, isopropyloxy group, n-butoxy group, sec-butoxygroup, t-butoxy group, n-hexyloxy group, 2-hexyloxy group or 3-hexyloxygroup; or

(c) a halogen atom, e.g., fluorine atom, chlorine atom, bromine atom oriodine atom, and

R¹ is

(a) a linear or branched lower alkyl group, e.g., methyl group, ethylgroup, propyl group, isopropyl group, n-butyl group, sec-butyl group,t-butyl group, n-hexyl group, 2-hexyl group or 3-hexyl group;

(b) a substituted cycloalkyl group, e.g., 2-phenyl-cycloalkyl group; or

(c) a substituted or unsubstituted aryl group, e.g.,4-chloro-3-methylphenyl group.

Preferable examples of the trans-3-substituted glycidic acid ester (I)are, for instance, compounds of the formula (I) wherein the ring A ismethylphenyl group or methoxyphenyl group and R¹ is methyl group, ethylgroup, 2-phenylcyclohexyl group or 4-chloro-3-methylphenyl group. Inparticular, the compounds of the formula (I) wherein the ring A is4-methylphenyl group or 4-methoxyphenyl group and R¹ is methyl group orethyl group are the more preferred examples.

Any ester residue may be used for the ester residue of the estercompound (B′) so long as it gives the ester compound (B′) a goodsolubility in the solvent used in the crystallization.

The ester residues of the ester compound (B′) are, for instance,

(a) a linear or branched alkyl group which may have a substituent andwhich has more carbon atoms than that of the ester residue R¹ of anoptical isomer (B), e.g., propyl group, isopropyl group, n-butyl group,sec-butyl group, t-butyl group, n-pentyl group, 2-pentyl group, 3-pentylgroup, n-hexyl group, 2-hexyl group, 3-hexyl group, n-heptyl group,2-heptyl group, 3-heptyl group, 4-heptyl group, n-octyl group, 2-octylgroup, 3-octyl group, 4-octyl group, n-nonyl group, 2-nonyl group,3-nonyl group, 4-nonyl group, 5-nonyl group, n-decyl group, 2-decylgroup, 3-decyl group, 4-decyl group or 5-decyl group;

(b) an alkoxyalkyl group which may have a substituent, e.g.,methoxymethyl group, ethoxymethyl group, propyloxymethyl group,methoxyethyl group, methoxypropyl group, methoxybutyl group, ethoxyethylgroup or propyloxypropyl group; and

(c) an arylalkyl group which may have a substituent, e.g., benzyl group,phenethyl group, phenylpropyl group or naphthylmethyl group.

The substituent for the linear or branched alkyl group (a) and thealkoxyalkyl group (b) includes, for instance, a halogen atom such asfluorine atom, chlorine atom, bromine atom or iodine atom. Thesubstituent for the arylalkyl group (c) includes, for instance, a linearor branched lower alkyl group, e.g., methyl group, ethyl group, propylgroup, isopropyl group, n-butyl group, sec-butyl group, t-butyl group,n-hexyl group, 2-hexyl group or 3-hexyl group; a linear or branchedlower alkoxy group, e.g., methoxy group, ethoxy group, propyloxy group,isopropyloxy group, n-butoxy group, sec-butoxy group, t-butoxy group,n-hexyloxy group, 2-hexyloxy group or 3-hexyloxy group; a halogen atom,e.g., fluorine atom, chlorine atom, bromine atom or iodine atom; and thelike.

Preferable examples of the ester residue of the ester compound (B′) are,for instance, isopropyl group, n-butyl group, sec-butyl group, t-butylgroup, n-pentyl group, n-hexyl group, n-octyl group, n-nonyl group,n-decyl group, benzyl group and phenethyl group, if the ester residue ofthe trans-3-substituted glycidic acid ester (I) is methyl group or ethylgroup.

Further, the absolute configuration of the ester compound (B′) may beeither one of (2R,3S) and (2S,3R).

If the (2R,3S)-ester compound (B′) is dissolved in the solution, the(2S, 3R)-isomer (A) can be crystallized to give crystals of high purityuntil the concentration of the (2S, 3R)-isomer (A) in the mother liquorbecomes very low as compared with conventional processes. On the otherhand, if the (2S,3R)-ester compound (B′) is dissolved in the solution,the (2R,3S)-isomer (A) can be crystallized to give crystals of highpurity until the concentration of the (2R, 3S)-isomer (A) in the motherliquor becomes very low as compared with conventional processes.Therefore, in either case, the desired product can be obtained in anextremely higher yield as compared with conventional processes.

Any solvent which can be used in recrystallization oftrans-3-substituted glycidic acid esters (I) may also be employed as thecrystallization solvent of the present invention. Examples of suchcrystallization solvents are, for instance, an alcohol solvent such asmethanol, ethanol, n-propanol, isopropanol or n-butanol; an ethersolvent such as diethyl ether, t-butyl methyl ether, diisopropyl ether,tetrahydrofuran or dioxane; an aromatic hydrocarbon solvent which may besubstituted by a halogen atom, such as benzene, toluene, xylene,chlorobenzene or dichlorobenzene; an aliphatic hydrocarbon solvent whichmay be substituted by a halogen atom, such as hexane, cyclohexane,n-heptane, n-octane, dichloromethane, chloroform, 1,2-dichloroethane orcarbon tetrachloride; an ester solvent such as methyl acetate or ethylacetate; and the like. The solvents may be used alone or in an admixturethereof.

A suitable solvent may be selected depending on the ester residues oftrans-3-substituted glycidic acid ester (I) and the ester compound (B′)and the substituent on the phenyl group of the ester (I). It ispreferable to use solvents in which the solubility of the optical isomer(A) of the trans-3-substituted glycidic acid ester (I) greatly variesdepending on the temperature and the solubility of the ester compound(B′) is far larger than that of the optical isomer (A). For example,when trans-3-(4-methoxyphenyl)glycidic acid methyl ester is used as theester (I) and n-butyl ester thereof is used as the ester compound (B′),methanol, ethanol, xylene and the like are preferable as the solvent fordissolving these compounds.

The suitable amount of the solvent can be determined within the rangethat the optical isomers (A) and (B) and the ester compound (B′) can bedissolved once and the isomer (A) can be crystallized by lowering thetemperature of the solution. Thus, a proper range of the amount ofsolvent can be experimentally found according to the kind of opticalisomers (A) and (B) and the ester compound (B′), proportion thereof,crystallization temperature and so on. In general, when solvents inwhich the solubility of the optical isomers (A) and (B) are large andthe solubility is largely changed according to the temperature change isused, it may be possible to decrease the amount of the solvent.

For example, in case that the following mixture oftrans-3-(4-methoxyphenyl)glycidic acid esters is dissolved in 100 ml ofxylene and the resulting solution is cooled to −10° C., only (2R, 3S)methyl ester can be efficiently crystallized from the solution even inthe range wherein the concentration of (2R, 3S) methyl ester in thesolution is smaller than that of (2S, 3R) methyl ester.

Mixture of the glycidic acid esters dissolved in 100 ml of xylene(2R,3S) methyl ester 49 g (2S,3R) methyl ester 14 g (2S,3R) n-butylester 39 g Composition of the glycidic acid esters remaining in thesolution after crystallization of (2R,3S) methyl ester (2R,3S) methylester  9 g (2S,3R) methyl ester no change (2S,3R) n-butyl ester nochange

The concentration of the optical isomer (A) in the solution prior to thecrystallization may vary depending on the kinds of the isomer (A) andsolvent, the temperature of the crystallization and the like, but it isusually from 0.5 to 4 moles/liter.

In a solution in the process of the present invention, the ratio of theoptical isomer (A)/the optical isomer (B) is required to be within arange in which the optical isomer (A) is more easily crystallized thanthe optical isomer (B), the crystallization of which is inhibited by theester compound (B′).

Even under the (A)/(B) ratio wherein the optical isomer (B) wouldprecipitate if the ester compound (B′) were not present, the opticalisomer (A) can be obtained as crystals by the process of the presentinvention.

Of course, the solution which contains the optical isomer (A) in anamount larger than the optical isomer (B) can be used and is preferredin the process of the present invention, since such an additional largeamount of the optical isomer (A) can also be obtained as the crystals inaddition to the crystals of the optical isomer (A) obtained by theinhibition of crystallization of isomer (B) due to the presence of estercompound (B′). Thus, it is possible to start the process of the presentinvention from a solution containing a larger amount of the isomer (A)than the isomer (B).

Moreover, the inhibitory effect of the ester compound (B′) oncrystallization of the optical isomer (B) increases according to theincrease in the ratio of the ester compound (B′)/the optical isomer (B),and the increased amount of the optical isomer (A) can be obtained.Thus, the higher the ratio of the ester compound (B′)/the isomer (B) is,the more preferable the process of the present invention is.

The ratios of these compounds in the initial solution for thecrystallization of the present invention vary depending on the kinds ofthe ester residues of the ester compound (B′) and the solvent used inthe crystallization. In general, the molar ratio of the optical isomer(A)/the optical isomer (B) is from about 4/6 to about 10/1, and themolar ratio of the ester compound (B′)/the optical isomer (B) is fromabout 5/3 to about 10/1. Preferably, the molar ratio of the isomer(A)/isomer (B) is from about 1/1 to about 4/1, and the molar ratio ofthe ester compound (B′)/isomer (B) is from about 2/1 to about 7.8/1.

In the process of the present invention, the crystallization can also becarried out from a solution containing other components than the isomers(A) and (B) and the ester compound (B′). For instance, in addition tothese three components, the solution may contain an optical isomer(ester compound (A′)) different from the optical isomer (A) only in theester residue. In case that the ester compound (A′) is contained in thesolution, the molar ratio of the ester compound (A′)/the isomer (A) ispreferably at most 9/35 of the molar ratio of the ester compound (B′)/the isomer (B), so that the inhibitory effect of the ester compound(A′) on the crystallization of isomer (A) can be minimized and theisomer (A) can be obtained in a large amount and in high purity.

That is to say, the ester compound (A′) may be present in the solutionfor the crystallization of the present invention so long as the opticalisomer (A) is only crystallized due to the inhibitory effect of theester compound (B′) on the crystallization of the optical isomer (B)though the crystallization of the optical isomer (A) may be inhibited bythe presence of the ester compound (A′).

When the inhibitory effect of the ester compound (B′) present in thesolution on the crystallization of the isomer (B) is larger than that ofthe ester compound (A′) on the crystallization of the isomer (A), theisomer (A) can be easily crystallized than the isomer (B), and theisomer (A) can be obtained by the process of the present invention.

In the process of the present invention, it is required that at leastthree isomers, namely the optical isomers (A) and (B) and the estercompound (B′), are present in the solution. In this respect, the processof the present invention is clearly different from a process A forpreferential crystallization from a solution containing only the isomers(A) and (B). Also, the process of the present invention takes advantageof the inhibitory effect of the ester compound (B′) on thecrystallization of the isomer (B) and the isolation of the desiredisomer (A) can be achieved by one crystallization-isolation procedure.In these respects, the process of the present invention is alsodifferent from the preferential crystallization process in which it isnecessary to repeat the following steps (i) and (ii):

(i) seeding crystals of the optical isomer (A) to a solution containingonly the isomers (A) and (B) to crystallize and isolate the opticalisomer (A) and

(ii) seeding crystals of the optical isomer (B) to the solutionresulting in the step (i) to crystallize and isolate the optical isomer(B).

Furthermore, the process of the present invention differs from thepreferential crystallization, wherein the crystallization of an opticalisomer must be carried out using a solution containing a larger amountof one optical isomer and a smaller amount of the other optical isomer.In contrast to this, according to the process of the present invention,the crystallization of an optical isomer (A) is possible in a wide rangefrom the optical isomer (A) >> the optical isomer (B) in the solution tothe optical isomer (A) < the optical isomer (B) therein.

Concomitantly, the process of the present invention includes theembodiment wherein the crystallization is carried out from a solution inwhich the crystals of the optical isomer (A) are also included. However,the crystallization of the optical isomer (A) from a solution notincluding at least one of the optical isomer (B) and the ester compound(B′) is excluded from the present invention because no inhibition of thecrystallization of the optical isomer (B) is possible.

The crystallization according to the process of the present inventionmust be carried out at a temperature at which the optical isomer (A) oftrans-3-substituted glycidic acid ester (I) is crystallized, but theoptical isomer (B) and the ester compound (B′) do not precipitate.

The temperature at which the crystallization of the optical isomer (B)begins only after the solution is allowed to stand for some time isincluded within the temperature range of the present invention. Suchcrystallization temperature varies depending on the kind oftrans-3-substituted glycidic acid ester (I), the kind of solvent and thecomposition of the solution to be subjected to crystallization.Considering the stability of the oxirane ring of trans-3-substitutedglycidic acid ester (I), it is not desirable to prepare thecrystallization solution by dissolving the optical isomers (A) and (B)and the ester compound (B′) at a higher temperature. It is preferablethat the dissolution is carried out at a temperature of not higher than70° C., and the crystallization is carried out at a temperature nothigher than room temperature.

Also, in general, when the amount of the solvent is large, thecrystallization of the isomer (A) does not proceed unless the solutionis cooled to a low temperature, while when the amount of the solvent issmall, the crystallization proceeds even at a relatively hightemperature. Therefore, when the process of the present invention iscarried out on an industrial scale, it is preferable from the viewpointsof the amount of the solvent, installation and energy that the amount ofthe solvent is decreased and the crystallization is carried out from aconcentrated solution at around room temperature.

On the other hand, when the crystallization is carried out from aconcentrated solution, the product usually tends to contain an increasedamount of impurities. Therefore, from the viewpoint of purity, it ispreferable to use a large amount of a solvent and to carry out thecrystallization at a low temperature.

For such reasons, in order to efficiently carry out the crystallizationof the optical isomer (A), an optimum temperature range should beexperimentally determined depending on the kind of trans-3-substitutedglycidic acid ester (I), the ester residue of the ester compound (B′),the kind of the solvent used and the composition of the solution to besubjected to crystallization.

For instance, when the optical isomers (A) and (B) is methyl esters andthe ester compound (B′) is a n-butyl ester and the crystallization iscarried out from methanol, it is preferable to carry out thecrystallization at a temperature of

30° to +15° C. A similar temperature range may be applied for othercases.

In the process of the present invention, since the crystallization ofthe optical isomer (B) is inhibited by the ester compound (B′), theoptical isomer (A) free from any contamination of the optical isomer (B)can be obtained without any very strict temperature control, which isnecessary for the preferential crystallization, taking advantage of thedifference in precipitation velocity between the optical isomers havingthe same solubility. Therefore, the allowable temperature range of thepresent invention is wider than the preferential crystallizationprocess.

According to the process of the present invention, the crystallizationof the optical isomer (B) is inhibited by the ester compound (B′). Whenprecipitation of the optical isomer (B) occurs, the optical isomer (B)precipitates together with the ester compound (B′) in an amorphous-likeform. The crystallization of the optical isomer (A) is visuallydistinguished from such precipitation.

Concomitantly, the crystallization of the optical isomer (A) accordingto the present invention can be carried out from a solution containingthe optical isomer (A) and (B) and the ester compound (B′) up to theextent that the optical isomer (B) would precipitate if the estercompound (B′) were not present. Since whether such extent is reached ornot depends on the solubility of the optical isomers (A) and (B) and theester compound (B′), even if the extent is not reached at a temperature,such extent may be reached at another temperature, and vice versa.

In the following lines, the condition under which the optical isomer (A)is crystallized from a solution containing the optical isomers (A) and(B) and the ester compound (B′), but the optical isomer (B) does notprecipitate by the presence of the ester compound (B′) though theoptical isomer (B) would precipitate in the absence of the estercompound (B′), is explained by the use of FIG. 1 illustrating the extentto be reached by the process of the present invention by the use of theparameters of temperature and time. In this connection, it is assumedthat the initial concentration of the optical isomer (A) in the solutionis larger than that of the optical isomer (B) and the amount of theester compound (B′) in the solution is sufficient for the inhibition ofthe crystallization of the optical isomer (B).

The solution (a) is a completely homogeneous solution. If the solution(a) is cooled, the isomer (A) begins to crystallize at the point (b). Ifthe cooling of the solution is further continued, the isomer (A) inexcess of the isomer (B) in the solution crystallizes to reach the point(c) at which the molar ratio of the isomer (A)/the isomer (B) is 1/1.

Theoretically, it is possible that only the isomer (A) is crystallizedduring the cooling of the solution (a), i.e., from the point (b) to thepoint (c). However, in a process disclosed in Japanese PatentPublication Kokai No. 8-259552, the isomer (A) is crystallized only tothe extent that the molar ratio of the isomer (A)/the isomer (B) in themother liquor after the crystallization is about 1.3/1. This is becauseaccording to knowledge of general crystallization technique, if thesolution (a) is cooled from the point (b) toward the point (c), theratio of the optical isomer (B)/the optical isomer (A) in the solutionincreases through the precipitation of the crystals of the opticalisomer (A) and the solubility of the optical isomer (B) decreases.Therefore, the fluctuation of the solution (i.e., partial non-uniformityof temperature, concentration and so on of the solution) exerts aninfluence on the crystallization of the isomer (B).

The solution at the point (c) contains isomers (A) and (B) in equalamounts. Therefore, when the solution is cooled from the point (c), theoptical isomers (A) and (B) were considered to crystallize in the formof the equimolar mixture thereof.

Nevertheless, in the present invention, since the ester compound (B′) ispresent in the solution, crystallization of the isomer (B) is inhibitedthereby. Thus, even if the solution is further cooled from the point(c), the isomer (A) is crystallized, but the isomer (B) is notcrystallized, so the yield of the isomer (A) can be increased.

After passing through the point (c), if the solution is further cooledto a lower temperature, the solution finally reaches the point (d) atwhich the whole solution becomes cloudy and not only the isomer (A) iscrystallized, but also an amorphous-like form of the isomer (B) and theester compound (B′) precipitates. Therefore, it is necessary to carryout the process of the present invention at a temperature higher thanthe point (d).

After passing through the point (c), if the solution is further cooled,for example, to reach the point (e) and the solution is maintained forsome time at that temperature, the solution reaches the point (f) afteran elapse of time t, where the whole solution becomes cloudy and thesame phenomenon as appearing at the point (d). Namely, the isomer (B)which has been inhibited from precipitation by the ester compound (B′)precipitates together with the ester compound (B′) as an amorphous-likeform. Therefore, the process of the present invention is required tofinish the crystallization and isolation of the optical isomer (A)before time t at which the solution becomes cloudy.

As explained above, the extent of crystallization aimed by the processof the present invention is within the region “the extent to be reachedby the process of the invention” shown in FIG. 1. This region variesdepending on various factors, e.g., the kind and ratio of the isomers(A) and (B) and the ester compound (B′), and the kind and amount of thesolvent. Thus, the suitable region can be determined in accordance withthese factors. For instance, in the case that the amount of the isomer(A) in the solution (a) as shown in FIG. 1 is smaller than that of theisomer (B), the starting point of the crystallization of the isomer (A)is somewhere between the points (d) and (c) and it is possible tocrystallize and isolate the isomer (A) in pure form up to the point (d).

The isolation of crystalline isomer (A) may be carried out in a usualmanner such as decantation and filtration. If the crystallization iscarried out in a large scale, a long time is required for the isolation,though the isolation in small scale can be accomplished in a short time.In case of the isolation on a laboratory scale, even immediate isolationis possible. If the isolation requires a long time as in the case ofindustrial production, it is preferable to interrupt thecrystallization, for instance, at the point (e), so that the isolationcan be completed without any contamination within the time length up tothe point t.

According to the process of the present invention, if thecrystallization is carried out, for instance, from a solution containingracemic trans-3-(4-methoxyphenyl)glycidic acid methyl ester and (2S,3R)-3-(4-methoxyphenyl)glycidic acid n-butyl ester, the amount of whichis sufficient to inhibit the precipitation of the (2S,3R)-isomer of themethyl ester, it is possible to crystallize (2R, 3S)-3-(4-.4-;methoxyphenyl)glycidic acid methyl ester until the concentration of (2S,3R)-3-(4-methoxyphenyl)glycidic acid methyl ester in the mother liquorafter the crystallization becomes at least twice as much as that of (2R,3S)-3-(4-methoxyphenyl)glycidic acid methyl ester. Further, it is alsopossible to obtain the crystals of the (2R,3S)-isomer having opticalpurity of at least 99%.

Thus, according to the present invention, after carrying out thecrystallization of the optical isomer (A) without any precipitation ofthe optical isomer (B) until the amount of the isomer (A) in the motherliquor is equal to that of the isomer (B), it is possible to continuethe crystallization of the isomer (A) in high purity until the amount ofthe isomer (A) in the mother liquor becomes smaller than that of theisomer (B).

Japanese Patent Publication Kokai No. 8-259552 discloses that crystalsof (2R,3S)-3-(4-methoxyphenyl)glycidic acid methyl ester having opticalpurity of at least 99% are obtained by asymmetrically transesterifyingracemic trans-3-(4-methoxyphenyl)glycidic acid methyl ester in thepresence of an esterase derived from Serratia marcescens to convert7.8/8.8 (about 88.6%) of the (2S,3R)-isomer into the n-butyl ester,removing the enzyme, distilling away the solvent under reduced pressure,and then conducting crystallization from isopropanol.

However, the crystallization is interrupted at the stage that the amountof the desired (2R,3S)-3-(4-methoxyphenyl)glycidic acid methyl ester inthe mother liquor after the crystallization is about 1.3 times as muchas that of (2S,3R)-3-(4-methoxyphenyl)glycidic acid methyl ester. Thisis because it was considered to be necessary to interrupt thecrystallization so as to avoid the contamination by the crystallizationof the undesired (2S, 3R)-isomer.

In contrast, according to the present invention, the crystallization canbe carried out until the amount of the desired (2R,3S)-3-(4-methoxyphenyl)glycidic acid methyl ester in the mother liquorafter the crystallization becomes about half of the undesired (2S,3R)-3-(4-methoxyphenyl)glycidic acid methyl ester.

The extent to which the isomer (A) is crystallized, namely the extent upto which the crystallization is continued, varies depending on the ratioof trans-3-substituted glycidic acid ester (I)/ester compound (B′),solvent for crystallization, crystallization temperature and the like.However, since the solution subjected to the crystallization becomescloudy at a stage that an amorphous-like form of the isomer (B) and theester compound (B′) begins to precipitate, it is possible to recognizethe end point of the crystallization of the isomer (A) by monitoring theappearance of cloudiness in the solution.

The purity of crystals may be generally affected by concentration of asolution to be subjected to crystallization (or the ratio of asolvent/the amount of the optical isomers), the ratio of optical isomer(A)/optical isomer (B), temperature, amount of crystals obtained and thelike.

Generally speaking, the purity of the crystals of the optical isomer (A)tends to be high if the crystallization is carried out up to the extentthat the isomers (A) and (B) are hard to be crystallized and the amountof the precipitated crystals of the optical isomer (A) is small. Forexample, if the concentration of the isomers (A) and (B) in thecrystallization solution is low, the ratio of the isomer (A)/the isomers(A) and (B) is high, the crystallization temperature is high and theamount of crystals of the optical isomer (A) is small, the purity of thecrystalline isomer (A) is high. In contrast to this, the yield of thecrystals of the optical isomer usually tends to be low if theconcentration is low.

However, according to the present invention, the precipitation of theoptical isomer (B) is inhibited on account of the presence of the estercompound (B′) and thus, it is possible to obtain optical isomer (A)having purity higher than 99% in a high yield.

The crystallization procedure is very simple since it comprises merelyconducting the crystallization of the optical isomer (A) from a solutionof the optical isomers (A) and (B) and the ester compound (B′) up to theextent that the optical isomer (B) does not precipitates due to thepresence of the ester compound (B′) though the optical isomer (B) wouldprecipitate in the absence of the ester compound (B′), thereby thecrystallization is continued up to the range where the amount of theoptical isomer (A) is smaller than that of the optical isomer (B) in themother liquor of the crystallization.

In conventional crystallization, crystals of the optical isomer (A) areobtained by crystallizing from a solution (AB) containing the opticalisomers (A) and (B) up to the extent that the amount of the isomer (A)in the mother liquor is larger than that of the isomer (B). In suchcrystallization, the solvent, temperature and the concentration werecarefully selected respectively in order to obtain the optical isomer(A) in high purity without any crystallization of the optical isomer(B), but the yield of the optical isomer (A) was obliged to besacrificed to some extent.

However, according to the process of the present invention, the opticalisomer (A) can be crystallized without precipitation of the isomer (B)even up to the extent that both of the optical isomers (A) and (B) wouldbe crystallized in the conventional crystallization wherein the estercompound (B′) were absent in the crystallization solution.

The preparation of the solution (ABB′) and the solution. (AA′BB′) to besubjected to the crystallization is explained below.

The ester compound (A′) may act to lower the yield of isomer (A) and,therefore, it is preferable that a solution to be subjected to thecrystallization does not contain the ester compound (A′). Since theester compound (A′) is not a component to be positively added to thesolution though such contamination may in some cases not be avoidable inpreparing the solution, the solution (ABB′) is more preferable than thesolution (AA′BB′).

The solution (ABB′) can be prepared, for instance, by adding the estercompound (B′) to a solution (AB) containing the optical isomers (A) and(B) or by transesterfying the optical isomer (B) in the solution (AB) toan ester compound (B′) in the presence of an enzyme having astereoselective transesterification ability by the use of an alcohol(R³—OH in which R³ is a linear or branched alkyl group which may besubstituted, an alkoxyalkyl group which may be substituted or anarylalkyl group which may be substituted).

In case of the enzymatic transesterification, the solution (ABB′) isobtained only when the stereoselectivity of an enzyme is 100%, and anenzyme having a stereoselectivity of less than 100% gives the solution(AA′BB′). Therefore, it is preferable to use an enzyme having a goodstereoselectivity.

The solution (AB) is usually obtained by a chemical synthesis since theproduct thereof is an equimolar mixture of the optical isomers with theexception of asymmetric synthesis.

The ester compound (B′) remains in a high concentration in a motherliquor from which the optical isomer (A) has been crystallized andisolated according to the present invention and can be taken outtherefrom if necessary. The ester compound (B′) can also be obtained byenzymatically and selectively hydrolyzing ester compound (A′) in amixture of ester compounds (Al) and (B′).

In case of preparing the solution (ABB′), the ester compound (B′) isusually added to a solution (AB) so that the resulting solution (ABB′)satisfies the condition that the isomer (A) is crystallized, but theisomer (B) is not crystallized by the presence of the ester compound(B′).

Solution (AB) to which ester compound (B′) is added, is not limited to asolution of an equimolar mixture of the optical isomers (A) and (B), andmay be a solution containing the isomers (A) and (B) in differentamounts. For example, such solution (AB) may be prepared by anasymmetric synthesis as disclosed, for instance, in Japanese PatentPublication Kokai No. 61-268663, No. 2-17170 and No. 2-17169, WO89/02428 and WO 89/10350.

The solution (AB) containing the isomers (A) and (B) in differentamounts may also be a solution prepared by subjecting a racemic solutionto an enzymatic and asymmetric hydrolysis disclosed in, for example,Japanese Patent Publication Kokai No. 2-109995 and No. 3-15398 andJapanese Patent Publication Kohyo No. 4-501360, or by subjecting aracemic solution to chemical optical resolution as disclosed for examplein Japanese Patent Publication Kokai No. 61-145174 and No. 2-231480.

As mentioned above, the process of the present invention may be carriedout in combination with known processes, e.g., asymmetric synthesis,chemical or enzymatic optical resolution and so on. In these cases, evenif the asymmetric induction or the rate of optical resolution in theknown processes is insufficient, the optical isomer (A) having a highpurity can be recovered in a good yield by combining such insufficientprocess with the process of the present invention.

Also, the process of the present invention may be applied to a solutionprepared by subjecting a solution (AB) to an enzymatictransesterification instead of adding ester compound (B′) to thesolution (AB). For example, the process of the present invention isapplicable to a solution. (ABB′) obtained in the process disclosed inJapanese Patent Publication Kokai No. 4-228095, No. 6-78790 and No.8-259552 wherein (2R,3S)-3-(4-methoxyphenyl)glycidic acid methyl esteris obtained by subjecting racemic trans-3-(4-methoxyphenyl)glycidic acidmethyl ester to enzymatic asymmetric transesterification with n-butanolto convert the (2S,3R)-isomer.

Various applications and modifications of the process of the presentinvention are possible. For example, both of the optical isomers (A) and(B) can be obtained in high purity by way of a recycling process whichcomprises:

(a) preparing a solution of one optical isomer (A) and the other opticalisomer (B) of the ester compound (I), both of which are the opticalisomers due to the asymmetric carbons at 2- and 3-positions, and anester compound (B′) which is different from the isomer (B) only in theester residue R¹,

(b) crystallizing the optical isomer (A) from the solution up to theextent that the optical isomer (A) is crystallized without theprecipitation of the optical isomer (B) due to the presence of the estercompound (B′) though the optical isomer (B) would precipitate if theester compound (B′) were not present,

(c) isolating the crystals of the optical isomer (A),

(d) chemically transesterfying the ester compound (B′) included in theresulting mother liquor together with the remaining optical isomers (A)and (B) so as to convert the ester compound (B′) into the optical isomer(B) followed by the crystallization and isolation of the isomer (B),

(e) adding a racemic trans-3-substituted glycidic acid ester (I) and anester compound (B′) to the resulting mother liquor to provide a solutionto be subjected to the crystallization of step (b), and

(f) repeating the above-mentioned steps (b), (c), (d) and (e) in thisorder.

In the recycling process, the concentration of the isomer (B) in thereaction mixture of the chemical transesterification in the step (d) ishigher than that of the isomer (A) and, therefore, only the isomer (B)can be obtained by conducting a conventional crystallization andisolation.

If an ester compound (A′) is added to the reaction mixture of thechemical transesterification prior to crystallizing the isomer (B) inthe step (d), the isomer (B) can be more efficiently crystallized by aninhibitory effect of the ester compound (A′) on the crystallization ofthe isomer (A). In case that the ester compound (A′) is added in thestep (d), such ester compound (A′) included in the resulting motherliquor is chemically transesterified to the isomer (A) in the step

It is preferable that the amount of the ester compound (A′) added in thestep (d) corresponds to that of the ester compound (B′) included in thesolution in the step (a). The amount of the ester compound (A′) variesdepending on the ester residues of the isomers (A) and (B) and the estercompound (A′) and the solvent used for the crystallization, but ingeneral the amount of the ester compound (A′) may be adjusted so thatthe molar ratio of ester compound (A′)/isomer (A) is from about 5/3 toabout 10/1.

The chemical transesterification adopted in the recycling process iscarried out by adding an organic amine and an alcohol corresponding tothe ester residue of an optical isomer to be obtained to the motherliquor obtained in the step (c), and conducting the transesterificationfollowed by distilling away the organic amine and the alcohol. Thechemical transesterification of the ester compound (A′) is carried outin the same manner.

The amount of the alcohol to be used in the chemical-transesterificationmay be preferably from 1 to 1,000 moles, especially 2 to 10 moles, permole of an ester to be transesterified. Alcohols having a low boilingpoint, e.g., methanol or ethanol can be preferably used for the chemicaltransesterification in view of the recycling of the isomers (A) and (B).

Examples of the amine used in the chemical transesterification are, forinstance, a monoalkylamine (e.g., methylamine, ethylamine, propylamine,butylamine); a dialkylamine (e.g., dimethylamine, diethylamine,dipropylamine, dilsopropylamine); a trialkylamine (e.g., trimethylamine,triethylamine); a cyclic amine (e.g., morpholine); and an aromatic amine(e.g., pyridine). Use of a dialkylamine having a low boiling point,e.g., dimethylamine, dipropylamine or diisopropylamine is particularlypreferred. The organic amine is preferably used in an amount of 0.01 to1,000 moles, especially 0.1 to 10 moles, per mole of the ester to betransesterified.

When the above recycling process is conducted, both of the isomers (A)and (B) can be obtained in high purity in one cycle. Since the processcan be practically carried out infinitely if the organic amine andalcohol can be sufficiently distilled away after the chemicaltransesterification, the isomers (A) and (B) having a high purity can beefficiently obtained only by recycling the process without conducting anasymmetric chemical reaction.

Moreover, the decrease in optical purity due to the contamination of theantipode on account of the fluctuation (partial nonuniformity insolution temperature, concentration and so on) of a crystallizationsolution can be suppressed and the amounts of the optical isomersobtained in one cycle is large. Therefore, the recycling process isindustrially advantageous.

A process wherein a solution containing optical isomers (A) and (B) andester compound (B′) is prepared by a transesterification process and isthen applied to the process of the present invention is explained below.

It is preferable to prepare a solution to be used for thecrystallization of optical isomer (A) by enzymatically transesterifyingthe isomer (B) in a solution of optical isomers (A) and (B) into theester compound (B′), since it is possible to obtain in a single reactionstep a solution containing a larger amount of the isomer (A) and asmaller amount of the isomer (B) together with the ester compound (B′)which inhibits crystallization of the isomer (B).

In the process of the present invention, it is preferable for the ratioof the isomer (A)/the isomer (B) in the reaction mixture resulting fromthe transesterification to be as large as possible. In other words, itis preferable for the ester compound (B′) to be abundant in the reactionmixture. In this case, in order to decrease the amount of the isomer (B)therein, it is necessary, for example, to increase the amounts of theenzyme and the alcohol used for the transesterification and to conductthe reaction for a longer time. However, such procedures increase thecost and cause side reactions.

According to the process of the present invention, even if the degree oftransesterification is low, the resulting solution may be successfullyused for the optical resolution unlike conventional processes. This isbecause that the ester compound (B′) inhibits precipitation of theisomer (B). The isomer (A) can be obtained in high purity in a highyield so long as the molar ratio of isomer (A)/isomer (B) is from 4/6 to10/1 and the molar ratio of ester compound (B′)/isomer (B) is from 5/3to 10/1. It is particularly preferable that the molar ratio of isomer(A)/isomer (B) is from 3/1 to 4/1 and the molar ratio of ester compound(B′)/isomer (B) is from 2/1 to 7.8/1.

The alcohols used for the transesterification are those whichcorresponds to the above-mentioned ester residue of the ester compound(B′).

Any enzymes having an ability of stereoselectively transesterifyingtrans-3-substituted glycidic acid ester (I) with the alcohol may be usedin the transesterification.

Such enzymes capable of selectively transesterifying the (2S, 3R)-isomerof the trans-3-substituted glycidic acid ester (I) include, forinstance, esterases derived from microorganisms belonging to the generaSerratia, Candida, Mucor, Pseudomonas, Aspergillus, Alcaligenes,Absidia, Fusarium, Giberella, Neurospora, Trichoderma, Rhizopus,Achromobacter, Bacillus, Brevibacterium, Corynebacterium, Providencia,Saccharomycopsis, Nocardia and Arthrobacter, α-chymotrypsin, porcineheper esterase, porcine pancreas esterase, and the like.

Representative examples of the above-mentioned enzyme are, for instance,esterases derived from Absidia corymbifera IFO 4009 and IFO 4010,Aspergillus ochraceus IFO 4346, Aspergillus terreus IFO 6123, Fusariumoxysporum IFO 5942, Fusarium oxysporum ATCC 659, Fusarium solani IFO5232, Gibberella fujikuroi IFO 5368, Mucor angulimacrosporus IAM 6149,Mucor circinelloides IFO 6746, Mucor flavus IAM 6143, Mucor fragilis IFO6449, Mucor genevensis IAM 6091, Mucor globosus IFO 6745, Mucorjanssenii IFO 5398, Mucor javanicus IFO 4569, IFO 4570, IFO 4572 and IFO5382, Mucor lamprosporus IFO 6337, Mucor petrinsularis IFO 6751, Mucorplumbeus IAM 6117, Mucor praini IAM 6120, Mucor pusillus IAM 6122, Mucorracemosus IFO 4581, Mucor ramannianus IAM 6128, Mucor recurvus IAM 6129,Mucor silvaticus IFO 6753, Mucor spinescens IAM 6071, Mucorsubtilissimus IFO 6338, Neurospora crassa IFO 6068, Rhizopus arrhizusIFO 5780, Rhizopus delemar ATCC 34612, Rhizopus japonicus IFO 4758,Trichoderma viride IFO 4847, Achromobacter cycloclastes IAM 1013,Bacillus sphaericus IFO 3525, Brevibacterium ketoglutamicum ATCC 15588,Corynebacterium alkanolyticum ATCC 21511, Corynebacteriumhydrocarboclastum ATCC 15592, Corynebacterium primorioxydans ATCC 31015,Providencia alcalifaciens JCM 1673, Pseudomonas mutabilis ATCC 31014,Pseudomonas putida ATCC 17426, ATCC 17453 and ATCC 33015, Serratialiquefaciens ATCC 27592, Serratia marcescens ATCC 13880, ATCC 14764,ATCC 19180, ATCC 21074, ATCC 27117 and ATCC_(21212,) Serratia marcescensSr4l FERM BP-487, Candida parapsilosis IFO 0585, Saccharomycopsislipolytica IFO 0717, IFO 0746, IFO 1195, IFO 1209 and IFO 1548, Nocardiaasteroides IFO 3384, IFO 3424 and IFO 3423, Nocardia gardneri ATCC 9604,Arthrobacter ureafaciens nov. var., Arthrobacter globiformis and Candidacylindracea.

Commercially available enzymes are also usable, e.g., alkaline lipase(from Achromobacter, Wako Pure Chemical Industries, Ltd.), Lipase B(from Pseudomonas fragi 22-39B, Wako Pure Chemical Industries, Ltd.),Lipase M AMANO” 10 (from Mucor javanicum, Amano Seiyaku KabushikiKaisha), Lipase type XI (from Rhizopus arrhizus, Sigma Chemical Co.,Ltd.), Talipase (from Rhizopus delemar, Tanabe Seiyaku Co., Ltd.),Lipase NK-116 (from Rhizopus japonicus, Nagase Sangyo Kabushiki Kaisha),Lipase N (from Rhizopus niveus, Amano Seiyaku Kabushiki Kaisha), LipaseSP 435 & 535 (from Candida antarctica, Novo), Alcalase (from Bacilluslicheniformis, Novo), Lipase type VII (from Candida cylindracea, SigmaChemical Co., Ltd.), lipase (from porcine pancreas, Wako Pure ChemicalIndustries, Ltd.), esterase (from porcine heper, Sigma Chemical Co.,Ltd.), cholesterol esterase (from Candida rugosa, Nagase SangyoKabushiki Kaisha), Lipase OF (from Candida cylindracea, Meito SangyoKabushiki Kaisha), Lipase QL (from Alcaligenes sp., Meito SangyoKabushiki Kaisha), Lipase AL (from Achromobacter sp., Meito SangyoKabushiki Kaisha), and Lipase PL (from Alcaligenes sp., Meito SangyoKabushiki Kaisha).

Among these enzymes, preferred are esterases derived from microorganismsbelonging to the genera Serratia, Candida, Alcaligenes andAchromobacter, particularly esterases derived from microorganisms suchas Serratia marcescens and Candida cylindracea

On the other hand, enzymes capable of selectively transesterifying the(2R, 3S)-isomer of the trans-3-substituted glycidic acid ester (I)include, for instance, esterases derived from microorganisms belongingto the genera Micrococcus, Agrobacterium, Microbacterium, Rhizobium,Citrobacter, Debaryomyces, Hanseniaspora, Hansenula, Pichia,Rhodosporidium, Schizosaccharomyces, Sporobolomyces, Kloeckera,Torulaspora and Streptomyces and the like.

Representative examples of such microbial enzymes capable of selectivelytransesterifying the (2R,3S)-isomer are, for instance, esterases derivedfrom Micrococcus ureae IAM 1010 (FERM BP-2996), Agrobacteriumradiobacter IAM 1526 and IFO 13259, Microbacterium sp. ATCC 21376,Rhizobium melioti IFO 13336, Citrobacter freundii ATCC 8090,Debaryomyces hansenii var. fabryi IFO 0015, Devaryomyces nepalensis IFO0039, Hanseniaspora valbyens IFO 0115, Hansenula polymorpha IFO 1024,Hansenula saturnus HUT 7087, Pichia farinosa IFO 0607, Pichia pastorisIFO 0948, IFO 1013 and IAM 12267, Pichia wickerhamii IFO 1278,Rhodosporidium toruloides IFO 0559, Schizosaccharomyces pombe IFO 0358,Sporobolomyces gracillis IFO 1033, Kloeckera corticis IFO 0633,Torulaspora delbrueckii IFO 0422, Streptomyces griseus subsp. griseusIFO 3430 and IFO 3355, and Streptomyces lavendulae subsp. lavendulae IFO3361, IFO 3415 and IFO 3146.

The enzymes used in the present invention may be commercially availableenzymes or enzymes obtained from a culture broth of microorganism cells.Also, the enzymes may be those obtained from wild strains or mutants, orthose obtained from microorganisms obtained by a biotechnologicaltechnique such as gene recombination or cell fusion.

The microbial enzymes as mentioned above can be obtained by culturing amicroorganism in a conventional manner, for instance, in a mediumcontaining usual carbon sources, nitrogen sources and inorganic salts atroom temperature or an elevated temperature under aerobic conditions atpH of 5 to 8, removing the cells from the culture broth in a usualmanner such as centrifugation or filtration, and optionally furtherremoving an impurity with a resin adsorbent. The culture broth of amicroorganism may be directly used as the enzyme.

The thus obtained solution may be directly used as the enzyme or may belyophilized. Also, the enzymes may be immobilized by a known method suchas a polyacrylamide method, a sulfur-containing polysaccharide gelmethod (e.g., carrageenin gel method), an agar gel method, aphoto-crosslikable resin method, a polyethylene glycol method, a Celitemethod or a membrane adsorption method. The immobilized enzymes may befilled in a column and used in the transesterification.

Meanwhile, enzymes with a high E-value have a high stereoselectivity.With such enzymes, optical isomer (A) is transesterified into estercompound (A′) in a less amount, while the optical isomer (B) istransesterified into ester compound (B′) in a larger amount. Since theamount of the ester compound (A′) which inhibits precipitation of thedesired isomer (A) must be decreased so as to increase the amount ofisomer (A) crystallized while the precipitation of isomer (B) isinhibited by the ester compound (B′), enzymes having a high E-value arepreferred among the enzymes as mentioned above.

The E-value is one of popular indications for representing thestereoselectivity of enzymes, and is an enzyme reaction velocity ratiowith respect to respective stereoisomers per unit concentration (e.g.,ratio of velocity of transesterification of the (2R, 3S)-isomer/that of(2S, 3R)-isomer).

In general, in case that the reaction has proceeded to a great degree,the E-value is strongly influenced by inactivation of enzyme, sidereaction, measurement error and the like. Thus, the E-value as usedherein is a value measured when the conversion rate is 10% (at the timewhen 10% of trans-dl-form has been transesterified) at which stage sidereaction, reverse reaction and measurement error are slight.

The E-value is represented by the following equation, and is a valueunique to each of enzymes.$E = \frac{\ln\left\lbrack {\left( {1 - c} \right)\left( {1 - {{ee}\left( {{optical}\quad {isomer}\quad A} \right)}} \right)} \right\rbrack}{\ln\left\lbrack {\left( {1 - c} \right)\left( {1 + {{ee}\left( {{optical}\quad {isomer}\quad A} \right)}} \right)} \right\rbrack}$wherein$c = \frac{{{amount}\quad {of}\quad {ester}\quad A^{\prime}} + {{amout}\quad {of}\quad {ester}\quad B^{\prime}}}{\begin{matrix}{{{amount}\quad {of}\quad {isomer}\quad A} + {{amount}\quad {of}\quad {isomer}\quad B} +} \\{{{amount}\quad {of}\quad {ester}\quad A^{\prime}} + {{amount}\quad {of}\quad {ester}\quad B^{\prime}}}\end{matrix}}$${{ee}\left( {{optical}\quad {isomer}\quad A} \right)} = \frac{{{amount}\quad {of}\quad {isomer}\quad A} - {{amount}\quad {of}\quad {isomer}\quad B}}{{{amount}\quad {of}\quad {isomer}\quad A} + {{amount}\quad {of}\quad {isomer}\quad B}}$

Thus, if the E-value and the transesterification conversion rate aredetermined, the ratio of optical isomers after the transesterificationcan be determined regardless of the kind of enzyme, and it is possibleto expect the optical purity and yield obtained therefrom.

The E-value preferable for the preparation of the solution to besubjected to the crystallization according to the present invention isat least 20, especially at least 50. Enzymes having a preferable E-valueas mentioned above can be suitably selected experimentally.

The amount of the enzyme varies depending on the kind of enzyme used,form of use and the like. But, it is desirable to use an enzyme at anamount having an olive oil hydrolysis activity of 1×10 to 1×10⁵U,especially 1×10² to 1×10⁴U, per g of isomer (B).

The olive oil hydrolysis activity is estimated by measuring the amountof the fatty acid generated by the cleavage of the ester bond in oliveoil by esterase according to the fat digesting power test reported inIyakuhin Kenkyu, vol. 11, No. 3, 505-506(1980).

The transesterification reaction can be carried out in an appropriatesolvent or in the absence of a solvent. Examples of the solvent are anaromatic organic solvent such as benzene, toluene or xylene; ahalogenated aromatic organic solvent such as chlorobenzene ordichlorobenzene; an aliphatic organic solvent such as hexane, heptane orcyclohexane; a halogenated aliphatic organic solvent such asdichloromethane, chloroform, carbon tetrachloride or trichloroethane; aketone solvent such as acetone, methyl ethyl ketone or methyl isobutylketone; an ether solvent such as dimethyl ether, diethyl ether,diisopropyl ether, tert-butyl methyl ether, tetrahydrofuran or1,4-dioxane; an ester solvent such as ethyl acetate or butyl acetate;and the like.

In particular, toluene, xylene, hexane, carbon tetrachloride, tert-butylmethyl ether and diisopropyl ether are preferred, since deactivation ofenzymes is small and the reaction proceeds in high velocity.

The concentration of the substrates, i.e., isomers (A) and (B), ispreferably from about 0.02 to about 3 moles/liter, especially about 0.02to about 2 moles/liter, since the reaction velocity is high and theinstallations for the transesterification reaction can be miniaturized.The amount of the alcohol for transesterification is preferably from 0.6to 10 moles, especially 0.8 to 5 moles, per mole of the isomer (B), sothat the enzyme can be prevented from deactivating and the reactionvelocity can be enhanced.

Contamination of the reaction system with water causes hydrolysis at thesame time of transesterification, thus resulting in decrease of yieldwhen the obtained solution is applied to the process of the presentinvention. Therefore, it is preferable to carry out the reaction withavoiding as much as possible the presence of extra water exceeding thatrequired for an enzyme to exhibit the transesterification activity.Further, the hydrolysis product is not soluble in some solvents and thisleads to decrease purity of the desired product due to the contaminationof such by-product.

The transesterification reaction is carried out at room temperature oran elevated temperature, preferably at a temperature of 10 to 50° C.,especially 20 to 40° C.

The conversion rate in the transesterification reaction can be suitablyselected in accordance with stereoselectivity of enzyme, yield ofoptical isomer (A) and the like. Such conversion rate can be freelyadjusted by changing enzyme activity, reaction temperature, reactiontime and the like in accordance with the substrate, trans-3-substitutedglycidic acid ester (I), and the ester residue of ester compound (B′).

In order to obtain optical isomer (A) of high purity in a high yield bya conventional process, it is generally required to raise the conversionrate in transesterification and to use an enzyme having very highselectivity.

However, in order to raise the conversion rate, at a stage that thereaction has proceeded to a great degree, the isomer (B) which remainsin a small amount in the reaction mixture must be furthertransesterified. Therefore, it is industrially impossible to raise theconversion rate to a high level considering the facts that a largeamount of expensive enzyme is necessary, the enzyme is easilydeactivated during the long time reaction, unstable trans-3-substitutedglycidic acid ester (I) is apt to be decomposed during the long reactionand some enzymatic transesterification of isomer (A) is inevitable dueto the insufficient selectivity of enzyme.

For example, Japanese Patent Publication Kokai No. 8-259552 disclosesthat asymmetric transesterification of racemictrans-3-(4-methoxyphenyl)glycidic acid methyl ester using esterasederived from Serratia marcescens is conducted to convert (2S, 3R)-isomerof the racemic methyl ester into (2S, 3R)-n-butyl ester until the molarratio of (2S,3R)-butyl ester/(2S,3R)-methyl ester becomes 7.8/1 [(2S,3R)-methyl ester 10.8 g and (2S, 3R) n-butyl ester 101.85 g].

However, this transesterification is carried out in such a manner thatthe reaction is firstly conducted for 24 hours using esterase of 5×10⁵Uper mole of the substrate, and after distilling away a solvent underreduced pressure until the volume of the reaction mixture becomes ⅓,esterase of 5×10⁵U is added and the reaction is further conducted for 16hours. Such a procedure is substantially inapplicable to industrialproduction, since the reaction time is too long, the procedure is toocomplicated and the amount of expensive enzyme is too large.

In contrast, according to the present invention, optical isomer (A) of ahigh purity can be obtained in a high yield even if the conversion ratein transesterification is not raised to a high level.

For example, (2R, 3S)-3-(4-methoxyphenyl)glycidic acid methyl esterhaving purity of 99% or more can be obtained in a yield of at least 80%from the reaction mixture obtained by the transesterification of racemictrans-3-(4-methoxyphenyl)glycidic acid methyl ester using esterasederived from Serratia marcescens Sr41 FERM BP-487 (1×10⁵U, 24 hours),wherein only 70% of the (2S, 3R)-isomer is converted into the n-butylester (ester compound B′/optical isomer B=7/3).

The present invention encompasses an embodiment wherein crystals of thesubstrate remain in the reaction mixture during the transesterificationreaction for the reason that the solubility of the substrate is low insome solvent.

In this case, after the transesterification reaction wherein theundesired isomer is converted into a soluble ester compound, the desiredproduct may be obtained from the reaction mixture by:

filtering the mixture of the enzyme and the desired isomer in crystalsand removing the enzyme contaminated in the desired isomer, while thefiltrate is cooled and the resulting crystals are obtained byfiltration, or

directly cooling the reaction mixture, filtering the mixture of theenzyme and the desired isomer in crystals and removing the enzymetherefrom.

The present invention also encompasses an embodiment wherein no crystalsprecipitate during the transesterification when the reaction isconducted in a normal procedure, but the crystals precipitate bypositively distilling away the solvent in the course of the reaction.The separation of the crystals precipitated by distillation of thesolvent can be conducted in the same manner as above. In case ofdistilling away a large portion of the solvent and adding a differentsolvent to the reaction mixture, the separation of the crystals can alsobe conducted in the same manner mentioned above.

In that case, an alcohol resulting from the transesterification can alsobe removed to the outside of the reaction system at the same time of thedistillation of the solvent and, therefore, the subsequenttransesterification comes to easily proceed.

As mentioned above, the process of the present invention is advantageousin that the optical isomer (A) of trans-3-substituted glycidic acidester (I) is preferably manufactured in industrial scale. This isbecause the isomer (A) can be obtained in high purity and in a highyield by coupling the crystallization and the transesterification, whichby itself was considered to be industrially inapplicable, even if thetransesterification of the racemate is stopped at a stage wherein theconversion rate is low.

The above explanation has been made with respect to a process whereinoptical isomer (A) is isolated by crystallizing the isomer (A) up to theextent that the isomer (B) is not crystallized due to the presence ofthe ester compound (B′) although optical isomer (B) would precipitate ifester compound (B′) were not present.

However, according to the process of the present invention, it is alsopossible to obtain the crystals of the optical isomer (A) in an amountlarger as compared with the conventional methods wherein the isomer (A)is crystallized from a solution (AB) under the condition that the isomer(A) is crystallized, but the isomer (B) is not. This is because theester compound (B′) in the solution (AA′BB′) or (ABB′) inhibits theprecipitation of the optical isomer (B) therefrom.

Moreover, the optical isomer (A) can be obtained in high purity and in ahigh yield in comparison of the conventional methods even from thesolution (ABB′) or the solution (AA′BB′) obtained by thetransesterification at low conversion rate.

For example, the isomer (A) of trans-3-substituted glycidic acid ester(I) is obtained in the form of crystals having optical purity of morethan 99% in a yield of more than 75% (in particular more than 80%) by

transesterifying the mixture of the isomers (A) and (B) with an alcoholcorresponding to the ester compound (B′) using an enzyme having anability of stereoselectively transesterifying the isomer (B) to theester compound (B′) (especially having the E-value of more than 20,e.g., esterase derived from Serratia marcescens Sr41 FERM BP-487) untilthe molar ratio of ester compound (B′)/isomer (B) becomes 13/7 to 7.8/1(especially 2/1 to 8/2) and

crystallizing the isomer (A) from the resulting reaction solution (thesolvent of which may be exchanged).

Concomitantly, the above-mentioned reaction solution, the solvent ofwhich is exchanged, includes, for instance, those obtained by partiallyor totally exchanging the solvent of the reaction solution so as toremove the resulting alcohol during the transesterification or bypartially or totally exchanging the solvent after thetransesterification so that the transesterification is carried out in asuitable solvent and the crystallization is accomplished in a propersolvent for that purpose.

The solution obtained by the transesterification may also be a solutionobtained by removing the enzyme from the reaction mixture resulting fromthe transesterification in a conventional manner such as filtration ordecantation.

From (2R,3S)-isomer of the trans-3-substituted glycidic acid ester,which is obtained above as the optical isomer (A), (2S,3S)-1,5-benzothiazepine derivatives or pharmaceutically acceptable saltscan be prepared by various processes, for example, by those disclosed inJapanese Patent Publication Kokoku No. 46-16749, Kokoku No. 63-13994,Kokai No. 5-201865, Kokai No. 2-289558 and Kokoku No. 2-28594, Chem.Pharm. Bull., 18(10), 2028-2037(1970), and Japanese Patent PublicationKokai No. 2-17168, No. 4-234866, No. 5-222016, No. 4-221376, No.5-202013, No. 2-17170, No. 2-286672, No. 6-279398, No. 58-99471, No.8-269026, No. 61-118377, No. 6-228117 and No.2-78673, European PatentPublication No. 796853 and Dutch Patent Publication No. 1006293.

Namely, (2S,3S)-1,5-benzothiazepine derivatives or pharmaceuticallyacceptable salts can be prepared by:

reacting the (2R,3S)-isomer with an aminothiophenol derivative of theformula (IV):

wherein the ring B is as defined above, and R4 is hydrogen atom,2-(dimethylamino)ethyl group or a group of the formula:

e.g., 2-aminothiophenol, 2-amino-5-chlorothiophenol,2-amino-5-benzylthiophenol, 2-(dimethylaminoethylamino)-thiophenol or acompound of the formula:

wherein the ring B is as defined above, or

reacting the (2R, 3S)-isomer with a nitrothiophenol derivative of theformula (V):

wherein the ring B is as defined above, e.g., 2-nitrothiophenol,2-nitro-5-chlorothiophenol or 2-nitro-5-benzylthiophenol, followed byreduction of the nitro group, to give a (2S,3S)-3-(2-aminophenylthio)-3-phenyl-2-hydroxypropionic acid ester of theformula (VI):

wherein the rings A and B, R¹ and R⁴ are as defined above,

subjecting the resulting compound (VI) to intramolecular ring closuredirectly or after conducting hydrolysis thereof, to give a (2S,3S)-2-phenyl-3-hydroxy-1,5-benzothiazepine derivative of the formula(VII):

wherein the rings A and B and R⁴ are as defined above, and

subjecting the resulting compound (VII) to dimethylaminoethylation atnitrogen atom of the 5-position and acetylation of hydroxyl groupsubstituted on the 3-position in arbitrary order to give a (2S,3S)-1,5-benzothiazepine derivative of the formula (II):

wherein the rings A and B and R² are as defined above, or apharmaceutically acceptable salt thereof.

The total reaction scheme of the above-mentioned conversion is shownbelow.

Examples of the (2S, 3S)-1,5-benzothiazepine derivatives (II) and thepharmaceutically acceptable salts thereof are, for instance, (2S,3S)-2-(4-methoxyphenyl)-3-acetoxy-5-[2-(dimethylamino)ethyl]-2,3-dihydro-1,5-benzothiazepin-4(5H)-one(diltiazem), (2S,3S)-2-(4-methoxyphenyl)-3-acetoxy-5-[2-(dimethylamino)ethyl]-8-chloro-2,3-dihydro-1,5-benzothiazepin-4(5H)-one, (2S,3S)-3-acetoxy-5-[3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl]-2,3-dihydro-2-(4-methoxyphenyl)-8-chloro-1,5-benzothiazepin-4(5H)-one,(2S,3S)-3-acetoxy-8-benzyl-2,3-dihydro-5-[2-(dimethylamino)ethyl)]-2-(4-methoxyphenyl)-1,5-benzothiazepin-4(5H)-one, and pharmaceutically acceptable salts thereof.

Meanwhile, from (2S, 3R)-isomer of the trans-3-substituted glycidic acidester, which is obtained above as the optical isomer (A),(2R,3R)-1,5-benzothiazepine derivatives or pharmaceutically acceptablesalts can be prepared in a similar manner to the above according to theknown processes.

Namely, (2R, 3R)-1,5-benzothiazepine derivatives or pharmaceuticallyacceptable salts can be prepared by:

reacting the (2S, 3R)-isomer with aminothiophenol derivative (IV), orreacting nitrothiophenol derivative (V) followed by reduction of thenitro group, in the same manner as the above-mentioned preparation of(2S,3S)-1,5-benzothiazepine derivatives (II) from the (2R, 3S)-isomer,to give a (2R, 3R)-3-(2-aminophenylthio)-3-phenyl-2-hydroxypropionicacid ester of the formula (VIII):

wherein ring A, ring B and R¹ are as defined above,

subjecting the resulting compound (VIII) to intramolecular ring closuredirectly or after conducting the hydrolysis thereof, to give a (2R,3R)-2-phenyl-3-hydroxy-1,5-benzothiazepine derivative of the formula(IX):

wherein ring A and ring B are as defined above, and

subjecting the resulting compound (IX) to dimethylaminoethylation atnitrogen atom of the 5-position and acetylation of hydroxyl groupsubstituted on the 3-position in arbitrary order to give a(2R,3R)-1,5-benzothiazepine derivative of the formula (III):

wherein ring A and ring B are as defined above, or a pharmaceuticallyacceptable salt thereof.

The total reaction scheme of the above-mentioned conversion is shownbelow.

Examples of the (2R, 3R)-1,5-benzothiazepine derivatives (III) and thepharmaceutically acceptable salts thereof are, for instance,(2R,3R)-cis-2-(4-methylphenyl)-3-acetoxy-5-[2-(dimethylamino)ethyl]-8-methyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one and pharmaceutically acceptable salts thereof.

Further, an optically active threo-nitrocarboxylic acid compound of theformula:

wherein ring A and ring B are as defined above and * denotes asymmetriccarbon atoms, which is useful as an optically resolving agent, can beprepared from the optical isomer (A) of trans-3-substituted glycidicacid ester obtained by the process of the present invention.

In the preparation of such optically active threo-nitrocarboxylic acidcompounds, the rings A and B are, for example, the same rings as thosementioned in the above-mentioned preparation of 1,5-benzothiazepinederivatives. Preferably, the ring A is a 4-lower alkoxyphenyl group andthe ring B is a substituted benzene ring of the formula:

wherein Hal is a halogen atom. More preferably, the ring A is4-methoxyphenyl group and the ring B is the substituted benzene ringshown by the above formula wherein Hal is chlorine atom.

The preparation of the threo-nitrocarboxylic acid compound can bepracticed, for example, by reacting the optical isomer (A) oftrans-3-substituted glycidic acid ester with a nitrothiophenol compoundof the formula:

wherein ring B is as defined above, in a manner as disclosed in JapanesePatent Publication Kokoku No. 61-18549, and then hydrolyzing the productaccording to a method disclosed in Chem. Pharm. Bull., 18(10),2028-2037(1970).

According to such processes, the (2S,3S)-isomer of thethreo-nitrocarboxylic acid compound can be obtained if the(2R,3S)-isomer of the trans-3-substituted glycidic acid ester (I) isused, and the (2R, 3R)-isomer of the threo-nitrocarboxylic acid compoundcan be obtained if the (2S, 3R) -isomer of the trans-3-substitutedglycidic acid ester (I) is used.

Concomitantly, the term “linear or branched lower alkyl group” as usedherein means a linear or branched alkyl group having 1 to 6 carbonatoms. Also, the term “linear or branched lower alkoxy group” as usedherein means a linear or branched alkoxy group having 1 to 6 carbonatoms. Further, the term “cycloalkyl group” as used herein means acycloalkyl group having 3 to 6 carbon atoms, and the term “aryl group”as used herein means an aryl group having 6 to 10 carbon atoms.

Also, the term “linear or branched alkyl group” as used herein means alinear or branched alkyl group having 1 to 12 carbon atoms. The term“alkoxyalkyl group” as used herein means an alkoxyalkyl group whereinthe alkoxy group has 1 to 6 carbon atoms and the alkyl group has 1 to 6carbon atoms. Also, the term “arylalkyl group” as used herein means anarylalkyl group wherein the aryl group has 6 to 10 carbon atoms and thealkyl group has 1 to 6 carbon atoms.

The present invention is more specifically described and explained bymeans of the following Examples. It is to be understood that the presentinvention is not limited to these Examples.

EXAMPLE 1

In 300 ml eggplant type flask were placed (2R,3S)-3-(4-methoxyphenyl)glycidic acid methyl ester (hereinafter referredto as “MPGN”), (2S, 3R)-MPGM and (2S, 3R)-MPGR³ [R³ ester of (2R,3S)-3-(4-methoxyphenyl)glycidic acid, i.e., a compound in which methylgroup of (2S, 3R)-MPGM was changed to R³ group; R³=n-propyl group,n-butyl group, n-octyl group, n-decyl group, benzyl group, cyclohexylgroup or 2-octyl group] in amounts shown in Table 1 together with 30 mlof methanol. They were dissolved in methanol at an elevated temperature.The solution was cooled to −15° C. with stirring by means of a magneticstirrer and allowed to stand at −15° C. for 5 minutes. Theconcentrations of the respective components in the resulting supernatantwere measured by HPLC*.

*: HPLC conditions: column, CHIRALCEL OD (Daicel Chemical Co.);Detection, UV at 237; Temp., 40° C.; Flow rate, 1.0 ml/min; Developingsolvent, Hexane/Isopropanol=20/1.

From the concentrations, the amounts of the respective componentsincluded in the supernatant were estimated. Further, the amount of thecrystals was estimated according to the equation: (amount of (2R,3S)-MPGM before crystallization)−(amount of (2R, 3S)-MPGM in thesupernatant).

The results are shown in Table 1.

Small portion of the crystals was obtained by filtration, washed withmethanol (−15° C. ) in an amount equal to the portion and dried invacuo, and the purity of the crystals was measured by HPLC. The contentof (2R,3S)-MPGM in the crystals was found to be 99% or more in allcases.

TABLE 1 Composition before Amount in Amount of crystals R³crystallization (g) supernatant (g) deposited (g) n-Propyl group(2R,3S)-MPGM: 9.8 (2R,3S)-MPGM: 1.1 (2R,3S)-MPGM: 8.7 (2S,3R)-MPGM: 2.0(2S,3R)-MPGM: no change (2S,3R)-MPGR³: 8.3 (2S,3R)-MPGR³: no changen-Butyl group (2R,3S)-MPGM: 9.9 (2R,3S)-MPGM: 1.0 (2R,3S)-MPGM: 8.9(2S,3R)-MPGM: 2.7 (2S,3R)-MPGM: no change (2S,3R)-MPGR³: 8.5(2S,3R)-MPGR³: no change n-Octyl group (2R,3S)-MPGM: 9.4 (2R,3S)-MPGM:1.4 (2R,3S)-MPGM: 8.0 (2S,3R)-MPGM: 2.2 (2S,3R)-MPGM: no change(2S,3R)-MPGR³: 6.7 (2S,3R)-MPGR³: no change n-Decyl group (2R,3S)-MPGM:10.3 (2R,3S)-MPGM: 1.6 (2R,3S)-MPGM: 8.7 (2S,3R)-MPGM: 2.8 (2S,3R)-MPGM:no change (2S,3R)-MPGR³: 8.6 (2S,3R)-MPGR³: no change Benzyl group(2R,3S)-MPGM: 9.6 (2R,3S)-MPGM: 1.6 (2R,3S)-MPGM: 8.0 (2S,3R)-MPGM: 2.2(2S,3R)-MPGM: no change (2S,3R)-MPGR³: 7.7 (2S,3R)-MPGR³: no changeCyclohexyl group (2R,3S)-MPGM: 9.7 (2R,3S)-MPGM: 1.5 (2R,3S)-MPGM: 8.2(2S,3R)-MPGM: 2.5 (2S,3R)-MPGM: no change (2S,3R)-MPGR³: 6.6(2S,3R)-MPGR³: no change 2-Octyl group (2R,3S)-MPGM: 9.3 (2R,3S)-MPGM:1.4 (2R,3S)-MPGM: 7.9 (2S,3R)-MPGM: 2.0 (2S,3R)-MPGM: no change(2S,3R)-MPGR³: 5.1 (2S,3R)-MPGR³: no change

EXAMPLE 2

In 300 ml eggplant type flask were placed (2R,3S)-MPGM, (2S,3R)-MPGM and(2S,3R)-MPGnBu [(2S,3R)-3-(4-methoxyphenyl)glycidic acid n-butyl ester]in amounts shown in Tables 2 to 7 together with 30 ml of methanol. Theywere dissolved by stirring the mixture with a 2.5 cm magnetic stirrer at300 r.p.m. The solution was cooled to a temperature shown in Tables 2 to7, and after the elapse of a predetermined time (0, 0.5 or 1 hour) thestirring was stopped. Small portion of the supernatant was taken out andthe concentrations of the respective components in the supernatant weremeasured by HPLC.

From the concentrations, the amounts of the respective componentsincluded in the supernatant were estimated. Further, the amount of thecrystals was estimated according to the equation: (amount of (2R,3S)-MPGM before crystallization)−(amount of (2R,3S)-MPGM in thesupernatant).

The clouding time denotes a time until a solution becomes cloudy afterreaching the crystallization temperature. The amounts of respectiveisomers in the supernatant were measured by HPLC with respect to samplestaken before occurrence of cloudiness.

TABLE 2 (2S,3R)-MPGnBu/(2S,3R)-MPGM = 1.7 by mole Composition beforeCrystallization Clouding time Amount in Amount of crystallization (g)temperature (° C.) (minute) supernatant (g) crystals (g) (2R,3S)-MPGM:10.3 10 >60 (2R,3S)-MPGM: 2.7 (2R,3S)-MPGM: 7.6 (2S,3R)-MPGM: 3.6(2S,3R)-MPGM: no change (2S,3R)-MPGnBu: 7.5 (2S,3R)-MPGnBu: no change

TABLE 3 (2S,3R)-MPGnBu/(2S,3R)-MPGM = 2.0 by mole Composition beforeCrystallization Clouding time Amount in Amount of crystallization (g)temperature (° C.) (minute) supernatant (g) crystals (g) (2R,3S)-MPGM:10.3 10 >60 (2R,3S)-MPGM: 2.5 (2R,3S)-MPGM: 7.8 (2S,3R)-MPGM: 3.4(2S,3R)-MPGM: no change (2S,3R)-MPGnBu: 7.9 (2S,3R)-MPGnBu: no change

TABLE 4 (2S,3R)-MPGnBu/(2S,3R)-MPGM = 2.2 by mole Composition beforeCrystallization Clouding time Amount in Amount of crystallization (g)temperature (° C.) (minute) supernatant (g) crystals (g) (2R,3S)-MPGM:10.3 10 >60 (2R,3S)-MPGM: 2.2 (2R,3S)-MPGM: 8.1 (2S,3R)-MPGM: 3.2(2S,3R)-MPGM: no change (2S,3R)-MPGnBu: 8.3 (2S,3R)-MPGnBu: no change(2R,3S)-MPGM: 10.3 0 35 (2R,3S)-MPGM: 1.6 (2R,3S)-MPGM: 8.7(2S,3R)-MPGM: 3.1 (2S,3R)-MPGM: no change (2S,3R)-MPGnBu: 8.2(2S,3R)-MPGnBu: no change (2R,3S)-MPGM: 10.3 −10 5 (2R,3S)-MPGM: 0.9(2R,3S)-MPGM: 9.4 (2S,3R)-MPGM: 3.1 (2S,3R)-MPGM: no change(2S,3R)-MPGnBu: 8.2 (2S,3R)-MPGnBu: no change

TABLE 5 (2S,3R)-MPGnBu/(2S,3R)-MPGM = 2.4 by mole Composition beforeCrystallization Clouding time Amount in Amount of crystallization (g)temperature (° C.) (minute) supernatant (g) crystals (g) (2R,3S)-MPGM:10.4 10 >60 (2R,3S)-MPGM: 2.2 (2R,3S)-MPGM: 8.2 (2S,3R)-MPGM: 3.0(2S,3R)-MPGM: no change (2S,3R)-MPGnBu: 8.7 (2S,3R)-MPGnBu: no change(2R,3S)-MPGM: 10.3 0 55 (2R,3S)-MPGM: 2.0 (2R,3S)-MPGM: 8.3(2S,3R)-MPGM: 2.9 (2S,3R)-MPGM: no change (2S,3R)-MPGnBu: 8.4(2S,3R)-MPGnBu: no change (2R,3S)-MPGM: 10.3 −10 50 (2R,3S)-MPGM: 1.1(2R,3S)-MPGM: 9.2 (2S,3R)-MPGM: 2.9 (2S,3R)-MPGM: no change(2S,3R)-MPGnBu: 8.4 (2S,3R)-MPGnBu: no change

TABLE 6 (2S,3R)-MPGnBu/(2S,3R)-MPGM = 2.7 by mole Composition beforeCrystallization Clouding time Amount in Amount of crystallization (g)temperature (° C.) (minute) supernatant (g) crystals (g) (2R,3S)-MPGM:10.3 10 >60 (2R,3S)-MPGM: 2.3 (2R,3S)-MPGM: 8.0 (2S,3R)-MPGM: 2.7(2S,3R)-MPGM: no change (2S,3R)-MPGnBu: 8.7 (2S,3R)-MPGnBu: no change(2R,3S)-MPGM: 10.3 0 >60 (2R,3S)-MPGM: 1.4 (2R,3S)-MPGM: 8.9(2S,3R)-MPGM: 2.7 (2S,3R)-MPGM: no change (2S,3R)-MPGnBu: 8.7(2S,3R)-MPGnBu: no change (2R,3S)-MPGM: 10.3 −10 55 (2R,3S)-MPGM: 0.8(2R,3S)-MPGM: 9.5 (2S,3R)-MPGM: 2.7 (2S,3R)-MPGM: no change(2S,3R)-MPGnBu: 8.7 (2S,3R)-MPGnBu: no change

TABLE 7 (2S,3R)-MPGnBu/(2S,3R)-MPGM = 3.8 by mole Composition beforeCrystallization Clouding time Amount in Amount of crystallization (g)temperature (° C.) (minute) supernatant (g) crystals (g) (2R,3S)-MPGM:10.3 0 >60 (2R,3S)-MPGM: 1.4 (2R,3S)-MPGM: 7.6 (2S,3R)-MPGM: 2.0(2S,3R)-MPGM: no change (2S,3R)-MPGnBu: 9.2 (2S,3R)-MPGnBu: no change(2R,3S)-MPGM: 10.3 −10 >60 (2R,3S)-MPGM: 0.8 (2R,3S)-MPGM: 9.5(2S,3R)-MPGM: 2.0 (2S,3R)-MPGM: no change (2S,3R)-MPGnBu: 9.2(2S,3R)-MPGnBu: no change

EXAMPLE 3

In 1,000 ml eggplant type flask were placed (2R,3S)-MPGM, (2S,3R)-MPGMand (2S,3R)-MPGnBu in amounts shown in Table 8 together with an amountof a solvent shown in Table 8. They were dissolved in the solvent bystirring the mixture with a 2.5 cm magnetic stirrer at 300 r.p.m. Thesolution was cooled to a temperature shown in Table 8, and immediatelythe stirring was stopped. Small portion of the supernatant was taken outand the concentrations of the respective components in the supernatantwere measured by HLPC.

From the concentrations, the amounts of the respective componentsincluded in the supernatant were estimated. Further, the amount of thecrystals was estimated according to the equation: (amount of (2R,3S)-MPGM prior to crystallization)−(amount of (2R, 3S)-MPGM in thesupernatant). The results are shown in Table 8.

Small portion of the crystals was obtained by filtration, washed withmethanol (−0° C. ) in an amount equal to the portion and dried in vacuo,and the purity of the crystals was measured by the use of HPLC. Thecontent of (2R,3S)-MPGM in the crystals was found to be 99% or more inall cases.

TABLE 8 Crystallization Amount of Crystallization Composition beforeAmount in Amount of solvent solvent (ml) temperature (° C.)crystallization (g) supernatant (g) crystals (g) Xylene 100 −10(2R,3S)-MPGM: 49 (2R,3S)-MPGM: 9 (2R,3S)-MPGM: 40 (2S,3R)-MPGM: 14(2S,3R)-MPGM: no change (2S,3R)-MPGnBu: 39 (2S,3R)-MPGnBu: no changeToluene 100 −10 (2R,3S)-MPGM: 49 (2R,3S)-MPGM: 9 (2R,3S)-MPGM: 40(2S,3R)-MPGM: 14 (2S,3R)-MPGM: no change (2S,3R)-MPGnBu: 39(2S,3R)-MPGnBu: no change Isopropyl ether 100 −10 (2R,3S)-MPGM: 49(2R,3S)-MPGM: 8 (2R,3S)-MPGM: 41 (2S,3R)-MPGM: 14 (2S,3R)-MPGM: nochange (2S,3R)-MPGnBu: 39 (2S,3R)-MPGnBu: no change t-Butyl 150 −10(2R,3S)-MPGM: 49 (2R,3S)-MPGM: 9 (2R,3S)-MPGM: 40 methyl (2S,3R)-MPGM:14 (2S,3R)-MPGM: no change ether (2S,3R)-MPGnBu: 39 (2S,3R)-MPGnBu: nochange Isopropanol 150 3 (2R,3S)-MPGM: 49 (2R,3S)-MPGM: 8 (2R,3S)-MPGM:41 (2S,3R)-MPGM: 14 (2S,3R)-MPGM: no change (2S,3R)-MPGnBu: 39(2S,3R)-MPGnBu: no change Ethanol 150 −10 (2R,3S)-MPGM: 50 (2R,3S)-MPGM:6 (2R,3S)-MPGM: 44 (2S,3R)-MPGM: 14 (2S,3R)-MPGM: no change(2S,3R)-MPGnBu: 42 (2S,3R)-MPGnBu: no change

EXAMPLE 4

In 300 ml eggplant type flask were placed (2R,3S)-MPGM, (2S,3R)-MPGM and(2S,3R)-MPGnBu in amounts shown in Table 9 together with methanol of anamount shown in Table 9. They were dissolved in methanol by stirring themixture with a 2.5 cm magnetic stirrer at 300 r.p.m. The solution wascooled to −10° C. . After stirring at that temperature for 1 hour,immediately the stirring was stopped. Small portion of the supernatantwas taken out and the concentrations of the respective components in thesupernatant were measured by HLPC.

From the concentrations, the amounts of the respective componentsincluded in the supernatant were estimated. Moreover, the crystals wereobtained by filtration, washed with methanol (−10C ) in an amount equalto the crystals and dried in vacuo, and the composition of the crystalswas measured by HPLC. The results are shown in Table 9.

TABLE 9 Amount Crystallization Composition before Amount in Amount ofComposition of solvent temperature crystallization supernatant crystalsobtained crystals (ml) (° C.) (g) (g) (g) (% by mole) 30 −10(2R,3S)-MPGM: 10.0 (2R,3S)-MPGM: 0.8 8.5 (2R,3S)-MPGM: 99.8(2R,3R)-MPGM: 2.5 (2R,3R)-MPGM: no change (2R,3R)-MPGM: 0.1(2R,3R)-MPGnBu: 9.3 (2R,3R)-MPGnBu: no change (2R,3R)-MPGnBu: 0.1 20 −10(2R,3S)-MPGM: 10.0 (2R,3S)-MPGM: 0.7 9.2 (2R,3S)-MPGM: 99.2(2R,3R)-MPGM: 2.5 (2R,3R)-MPGM: no change (2R,3R)-MPGM: 0.3(2R,3R)-MPGnBu: 9.3 (2R,3R)-MPGnBu: no change (2R,3R)-MPGnBu: 0.5

EXAMPLE 5

In 300 ml eggplant type flask were placed (2R, 3S)-MPGM, (2S, 3R)-MPGM,(2R, 3S)-MPGnBu and (2S, 3R)-MPGnBu in amounts shown in Table 10together with 30 ml of methanol. They were dissolved in methanol bystirring the mixture with a 2.5 cm magnetic stirrer at 300 r.p.m. Thesolution was cooled to −15° C. over 30 minutes. After further stirringat that temperature for 2 hours, the stirring was stopped, and smallportion of the supernatant was taken out and the concentrations of therespective components in the supernatant were measured by HLPC.

From the concentrations, the amounts of the respective componentsincluded in the supernatant were estimated. Further, the amount of thecrystals was estimated according to the equation: (amount of (2R,3S)-MPGM prior to crystallization)−(amount of (2R, 3S)-MPGM in thesupernatant).

Moreover, the crystals were obtained by filtration, washed with methanol(−15° C. ) in an amount equal to the crystals and dried in vacuo, andthe composition of the crystals was measured by HPLC. The results areshown in Table 10.

TABLE 10 Composition before Amount of Composition of (2R,3S)-MPGnBu(molar crystallization crystals deposited crystals (2R,3S)-MPGM ratio)(g) (g) (% by mole) 0.01 (2R,3S)-MPGM: 9.9 (2R,3S)-MPGM: 8.9(2R,3S)-MPGM: 99.7 (2S,3R)-MPGM: 2.7 (2S,3R)-MPGM: 0.1 (2R,3S)-MPGnBu:0.1 (2R,3S)-MPGnBu: 0.1 (2S,3R)-MPGnBu: 8.5 (2S,3R)-MPGnBu: 0.1 0.10(2R,3S)-MPGM: 8.1 (2R,3S)-MPGM: 6.5 (2R,3S)-MPGM: 96.1 (2S,3R)-MPGM: 2.1(2S,3R)-MPGM: 0.5 (2R,3S)-MPGnBu: 1.0 (2R,3S)-MPGnBu: 2.5(2S,3R)-MPGnBu: 5.5 (2S,3R)-MPGnBu: 0.9 0.25 (2R,3S)-MPGM: 7.6(2R,3S)-MPGM: 6.0 (2R,3S)-MPGM: 96.6 (2S,3R)-MPGM: 2.1 (2S,3R)-MPGM: 0.2(2R,3S)-MPGnBu: 2.3 (2R,3S)-MPGnBu: 2.9 (2S,3R)-MPGnBu: 7.1(2S,3R)-MPGnBu: 0.3 0.50 (2R,3S)-MPGM: 6.5 (2R,3S)-MPGM: 5.0(2R,3S)-MPGM: 93.8 (2S,3R)-MPGM: 1.7 (2S,3R)-MPGM: 0.2 (2R,3S)-MPGnBu:3.8 (2R,3S)-MPGnBu: 5.5 (2S,3R)-MPGnBu: 9.0 (2S,3R)-MPGnBu: 0.5

EXAMPLE 6

A solution of 10.4 g of (2R,3S)-MPGM, 10.4 g of (2S,3R)-MPGM and 40.5 gof (2S,3R)-MPGnBu in 150 ml of methanol was placed in a 500 ml eggplantflask and was stirred at 300 r.p.m. by a 2.5 cm magnetic stirrer. Thesolution was cooled to −10° C. over 30 minutes and further stirred for10 minutes. The crystals were obtained by filtration, washed with 10 mlof methanol (−10° C. ) and dried in vacuo. The composition of thecrystals was measured by HPLC. The content of (2R, 3S)-MPGM in 4.5 g ofcrystals (yield of 43% based on the amount of (2R, 3S)-MPGM prior to thecrystallization) was found to be 99% or more.

After obtaining the crystals, 10 ml of methanol, which corresponds tothe washing liquid in the mixture of the filtrate and the washings, wasdistilled away.

The concentrated mixture was cooled to −10° C., and thereto were addedseed crystals of (2S, 3R)-MPGM. When the mixture was stirred at thattemperature for 30 minutes, it became cloudy and an amorphous-likematerial precipitated.

The amorphous-like material was obtained by filtration and thecomposition thereof was measured by HPLC. It was found that theamorphous-like material is a solid containing (2S, 3R)-MPGM having thesame configuration as the seed crystals in the largest amount togetherwith almost the same amount of (2R, 3S)-MPGM and about two-third amountof (2S,3R)-MPGnBu.

EXAMPLE 7

In a mixed solvent of 500 ml of xylene, 80 ml of n-butanol and 0.25 mlof water was dissolved 104 g (0.5 mole) of (2RS,3SR)-MPGM [52 g of(2R,3S)-MPGM and 52 g of (2S, 3R)-MPGM]. In the resulting solution wassuspended 200 mg of esterase derived from Serratia marcescens (olive oilhydrolysis activity: 4×10⁴U) which was obtained in Reference Example1-(2-b) described after. The solution was stirred at 30° C. for 24hours, and the reaction mixture was analyzed by HPLC. It was found thatthe product was composed of 50 g of (2R,3S)-MPGM, 13.5 g of (2S,3R)-MPGMand 44 g of (2S,3R)-MPGnBu. The enzyme was removed from the reactionmixture by filtration, and the whole solvent was distilled away from thefiltrate under reduced pressure to give an oily residue. To the residuewas added 150 ml of methanol, and it was stirred for crystallization. Inorder to further raise the yield, the mixture was cooled to −15° C. andstirred at the same temperature for 30 minutes. The crystals wereobtained by filtration, washed with 40 ml of methanol (-15° C. ) anddried in vacuo to give 44.2 g of (2R,3S)-MPGM.

From the analysis of the filtrate obtained at that time, it was alsofound that the mother liquor contained 6 g of (2R,3S)-MPGM, 13.5 g of(2S,3R)-MPGM and 44 g of (2S, 3R)-MPGnBu. The yield of the crystals was85.0% (percentage on the basis that (2R,3S)-MPGM in the charged racemiccompound was 100%). The content of (2R, 3S)-MPGM in the crystals wasfound to be 99% or more.

EXAMPLE 8

20.8 g of (2RS,3SR)-MPGM [10.4 g of (2R,3S)-MPGM and 10.4 g of(2S,3R)-MPGM], 100 ml of xylene, 16 ml of n-butanol, 25 μl of water and10 mg of esterase derived from Serratia marcescens (olive oil hydrolysisactivity: 2×10³U) which was obtained in Reference Example 1-(2-b)described after were mixed. The mixture was subjected to an enzymatictransesterification reaction at 30° C. until reaching thetransesterification conversion shown in Table 11. The enzyme was removedfrom the reaction mixture by filtration, and the whole solvent wasdistilled away from the filtrate at a temperature of 60 to 70° C. undera reduced pressure to give an oily residue.

To the residue was added 30 ml of methanol, and it was stirred in aneggplant type flask at 300 r.p.m. with a 2.5 cm magnetic stirrer. Aftercooling the mixture to −10° C. , the stirring was stopped. Small portionof the supernatant was taken out and the concentrations of thecomponents in the supernatant were measured by HLPC. From theconcentrations, the amounts of the compounds included in the supernatantwere estimated.

Further, the amount of the crystals was estimated according to theequation: (amount of (2R,3S)-MPGM prior to crystallization)−(amount of(2R, 3S)-MPGM in the supernatant). The results are shown in Table 11.

Small portion of the crystals was obtained by filtration, washed withmethanol of −10° C. in an amount equal to the crystals, dried in vacuoand analyzed by HPLC. The content of (2R, 3S)-MPGM in the crystals wasfound to be 99% or more in all cases.

TABLE 11 Composition before Amount in Amount of crystallizationsupernatant crystals deposited Conversion (g) (g) (g) 0.36 (2R,3S)-MPGM:10.2 (2R,3S)-MPGM: 0.9 (2R,3S)-MPGM: 9.3 (2S,3R)-MPGM: 3.1 (2S,3R)-MPGM:no change (2R,3S)-MPGnBu: 0.03 (2R,3S)-MPGnBu: no change (2S,3R)-MPGnBu:8.2 (2S,3R)-MPGnBu: no change 0.37 (2R,3S)-MPGM: 10.2 (2R,3S)-MPGM: 1.1(2R,3S)-MPGM: 9.1 (2S,3R)-MPGM: 2.9 (2S,3R)-MPGM: no change(2R,3S)-MPGnBu: 0.03 (2R,3S)-MPGnBu: no change (2S,3R)-MPGnBu: 8.3(2S,3R)-MPGnBu: no change 0.38 (2R,3S)-MPGM: 10.2 (2R,3S)-MPGM: 0.8(2R,3S)-MPGM: 9.4 (2S,3R)-MPGM: 2.7 (2S,3R)-MPGM: no change(2R,3S)-MPGnBu: 0.03 (2R,3S)-MPGnBu: no change (2S,3R)-MPGnBu: 8.6(2S,3R)-MPGnBu: no change 0.41 (2R,3S)-MPGM: 10.2 (2R,3S)-MPGM: 0.8(2R,3S)-MPGM: 9.4 (2S,3R)-MPGM: 2.0 (2S,3R)-MPGM: no change(2R,3S)-MPGnBu: 0.03 (2R,3S)-MPGnBu: no change (2S,3R)-MPGnBu: 9.2(2S,3R)-MPGnBu: no change

EXAMPLE 9

In a mixed solvent of 500 ml of xylene and 80 ml of n-butanol wasdissolved 104 g (0.5 mole) of (2RS,3SR)-MPGM [52 g of (2R,3S)-MPGM and52 g of (2S, 3R)-MPGM]. In the resulting solution was suspended 3.0 g ofesterase immobilized on Celite (olive oil hydrolysis activity: 2.5×10⁴U)which was obtained in Reference Example 1-(2-a) described after. Thesolution was stirred at 30° C. for 24 hours, and the reaction mixturewas analyzed by HPLC. It was found that the product was composed of 51 gof (2R,3S)-MPGM, 14.7 g of (2S,3R)-MPGM and 38 g of (2S,3R)-MPGnBu. TheCelite-immobilized esterase was removed from the reaction mixture byfiltration, and the whole solvent was distilled away from the filtrateat a temperature of 60 to 70° C. under reduced pressure to give an oilyresidue. To the residue was added 150 ml of methanol, and it was stirredfor crystallization. In order to further raise the yield, the mixturewas cooled to −10° C. and stirred at the same temperature for 30minutes. The crystals were obtained by filtration, washed with 40 ml ofmethanol of -10C and dried in vacuo to give 43.1 g of (2R,3S)-MPGM.

From the analysis of the filtrate obtained at that time, it was alsofound that the mother liquor contained 7 g of (2R,3S)-MPGM, 14.7 g of(2S,3R)-MPGM and 38 g of (2S,3R)-MPGnBu. The yield of the crystals was82.9% (percentage on the basis that (2R,3S)-MPGM in the charged racemiccompound was 100%). The content of (2R,3S)-MPGM in the crystals wasfound to be 99% or more.

EXAMPLE 10

(1) In 150 ml of methanol were dissolved 20 g of (2RS,3SR)-MPGM and 41 gof (2S,3R)-MPGnBu at a temperature of 30 to 40C . The solution wascooled with stirring, and 10 mg of seed crystals of (2R,3S)-MPGM wasadded to the solution at 0° C. The solution was further cooled to −10°C. over 30 minutes and was stirred at that temperature for 10 minutes.The crystals were obtained by filtration with a glass filter, washedwith 20 ml of methanol of −10° C. and dried under vacuum to give (2R,3S)-MPGM. The filtrate and washings were combined to give a methanolsolution containing (2R,3S)-MPGM, (2S,3R)-MPGM and (2S,3R)-MPGnBu.

(2) To the above methanol solution were added 100 ml of methanol and 10ml of diisopropylamine. The solution was stirred at 30° C. for 16 hoursto convert (2S,3R)-MPGnBu into (2S,3R)-MPGM.

(3) The reaction mixture obtained in the step (2) was allowed to standat 0° C. for 30 minutes. The crystals of (2S,3R)-MPGM were obtained byfiltration and washed with 40 ml of methanol of 5° C. The filtrate wasconcentrated under reduced pressure to give a residue containing (2S,3R)-MPGM and (2R, 3S)-MPGM in approximately equal amounts.

(4) To the residue was added (2RS,3SR)-MPGM so that the total amount of(2RS, 3SR)-MPGM became about 20 g. Thereto were further added (2S,3R)-MPGnBu and methanol. The resulting solution was applied to the abovestep (1). The procedure consisting of the steps (1) to (4) was repeatedthree times, and the results thereof are shown in Table 12.

TABLE 12 (Change in composition of components in methanol solution)Procedure (2R,3S)-MPGM (g) (2S,3R)-MPGM (g) (2S,3R)-MPGnBu (g) Additionof 10(+10 addition) 10(+10 addition) 41(+41 addition) racemic compoundCrystallization 4.4(−5.6 deposition) 10 41 Chemical 4.4 44 0(−41transesterification) transesterification (+34.1 transesterification−0.1*) Crystallization 3.6(−0.8***) 3.9(−37.9 deposition −2.2**) 0Addition of 10(+6.4 addition) 10.3(+6.4 addition) 41(+41 addition)racemic compound Crystallization 4.2 (−5.8 deposition) 10.3 41 Chemical4.2 44(+34.1 transesterification −0.4*) 0(−41 transesterification)transesterification Crystallization 4.2 4.4(−35.8 deposition −3.8**) 0Addition of 10(+5.8 addition) 10.2(+5.8 addition) 41(+41 addition)racemic compound Crystallization 5.2(−4.8 deposition) 10.2 41 Chemical5.2 44 0(−41 transesterification) transesterification (+34.1transesterification −0.3*) Crystallization 5.2 6.2(−37.0 deposition−0.8**) 0 *Loss at the time of transesterification **Loss at the time ofwashing crystals ***Loss caused by washing and so on

There was obtained 16.2 g of (2R,3S)-MPGM in total by the above threecycle procedure. The purity and optical purity of (2R,3S)-MPGM obtainedin each cycle were 98% or more and 97% or more, respectively.

Since (2S,3R)-MPGM can be obtained as crystals in a larger, molar amountthan (2S,3R)-MPGnBu used as an ester compound, it is also possible toobtain (2S, 3R)-MPGM having high purity if thus-obtained (2S, 3R)-MPGMis chemically transesterified to (2S, 3R)-MPGnBu only in an amountnecessary for (2S, 3R)-MPGnBu in the next cycle.

EXAMPLE 11

In a 2 liter reactor equipped with a stirrer, 187 g (0.9 mole) of(2RS,3SR)-MPGM [93.5 g of (2R,3S)-MPGM and 93.5 g of (2S,3R)-MPGM] wasdissolved in a mixed solvent of 720 ml of xylene and 57.6 ml (0.9 mole)of n-butanol. In the resulting solution was suspended 3.0 g ofCelite-immobilized esterase (to which 0.81 ml of purified water waspreviously added and which had an olive oil hydrolysis activity of2.5×10⁴U) which was obtained in Reference Example 1-(2-a) describedafter. The solution was stirred at 30° C. for 4 hours under a reducedpressure of 15 mmHg, and the reaction mixture was analyzed by HPLC. Itwas found that the product was composed of 91.6 g of (2R,3S)-MPGM, 24.3g of (2S,3R)-MPGM and 83.2 g of (2S,3R)-MPGnBu.

The Celite-immobilized esterase was removed from the reaction mixture byfiltration, and the filtrate was concentrated at a temperature of 60 to70° C. under reduced pressure to give an oily residue. To the residuewas added 150 ml of methanol, and it was cooled to −10° C. with stirringand further stirred at that temperature for 30 minutes. The crystalswere obtained by filtration, washed with methanol of −10° C. and driedin vacuo to give 82.3 g of (2R, 3S)-MPGM. The mother liquor and washingswere combined and analyzed by HPLC. It was found that the mixturecontained 9.1 g of (2R,3S)-MPGM, 24.1 g of (2S,3R)-MPGM and 83.0 g of(2S,3R)-MPGnBu.

REFERENCE EXAMPLE 1

(1) In a 30 liter jar fermenter was placed 18 liters of a liquid mediumof pH 7.0 containing 1% of dextrin, 0.2% of ammonium sulfate, 2% ofMeast S (made by Asahi Breweries, Ltd.), 0.2% of potassiumdihydrogenphosphate, 0.05% of magnesium sulfate, 0.001% of ferroussulfate, 1.5 w/v % of a sorbitane trioleate surfactant (Rheodol SP-030,made by Kao Corporation) and 0.2 v/v % of a polyalkylene glycolderivative surfactant (trade mark Kararin 102, made by Sanyo ChemicalIndustries, Ltd.). After sterilizing the medium, 200 ml of apre-cultured broth of Serratia marcescens Sr41 FERM BP-487 obtained bypreviously culturing with reciprocal shaking at 27.5° C. for 20 hours inthe same culture medium as above was inoculated into the sterilizedmedium. The mixture was cultured at 25° C. for 28 hours withcontinuously adding 1.5% of L-proline to the medium under the conditionsof 0.33 vvm, 0.5 kg/cm²·G and 300 r.p.m. Ten liters of the culture brothwas centrifuged to remove the cells, and impurities were removed fromthe supernatant by the use of an adsorptive resin SP207 (made byMitsubishi Chemical Corporation). The thus obtained supernatant wasconcentrated to 1 liter by using a ultrafiltration membrane (SLP1053made by Asahi Chemical Industry Co., Ltd.) to give 1,080 ml of aconcentrated liquid of esterase having an olive oil hydrolysis activityof 1.0×10⁴U/ml.

(2-a) After impregnating 30 ml of the concentrated enzyme liquidobtained in (1) into 30 g of Celite (made by Celite Corporation,California, U.S.A.) placed in a 500 ml eggplant type flask, they wereuniformly mixed and dried at an external temperature of 30° C. underreduced pressure by using a rotary evaporator to give 36.5 g ofCelite-immobilized esterase having an activity of 8.2 U/mg.

(2-b) Lyophilized was 1,000 ml of the concentrated enzyme liquidobtained in (1) to give 54 g of esterase having an olive oil hydrolysisactivity of 1.96×10⁵U/g.

REFERENCE EXAMPLE 2

A mixture of 225 ml of a 2% aqueous solution of polyvinyl alcohol (trademark Poval 117, made by Kuraray Co., Ltd.) and 75 ml of olive oil wasstirred at a temperature of 5 to 10° C. for 10 minutes at 14,500 r.p.m.to form an emulsion. Then, 5.0 ml of the obtained olive oil emulsion and4.0 ml of a 0.25 M tris-HCl buffer (pH8.0, containing 2.5 mM of calciumchloride) were pre-heated at 37° C. for 10 minutes, and thereto wasadded 1 ml of an enzyme liquid. The reaction was conducted at 37° C. for20 minutes, and 20 ml of an acetone-ethanol mixed solvent (1:1) wasadded to the reaction mixture to terminate the reaction. The reactionmixture was titrated with a 0.05 N NaOH solution using phenolphthaleinas the indicator. The amount of enzyme which liberated 1 μmole of afatty acid per minute by the above procedure was defined as one unit(U).

As explained above, according to the present invention, from a solutioncontaining a mixture of optical isomers of trans-3-substituted glycidicacid esters, a desired optical isomer having high purity can becrystallized until the concentration of the desired optical isomer inthe mother liquor becomes very low as compared with conventionalprocesses.

Further, after asymmetrically and enzymatically transesterifying racemictrans-3-substituted glycidic acid esters, a desired optical isomerhaving high purity can be crystallized from the reaction mixture untilthe concentration of the desired optical isomer in the mother liquorbecomes very low as compared with conventional processes.

What we claim is:
 1. In a process for preparing a(2S,3S)-1,5-benzothiazepine derivative of the formula (II):

wherein ring A and ring B are independently a substituted orunsubstituted benzene ring, and R² is a 2-(dimethylamino)ethyl group ora group of the formula:

or a pharmaceutically acceptable salt thereof, the improvementcomprising using as the starting material a (2R,3S)-isomer oftrans-3-substituted glycidic acid ester compound of the formula (I):

wherein ring A is a substituted or unsubstituted benzene ring, and R¹ isan ester residue, which is obtained by preparing a solution of the(2R,3S)-isomer (A) and the (2S,3R)-isomer (B) of the ester compound (I),both of which are optical isomers due to the asymmetric carbons at the2- and 3-positions, and an ester compound (B′) which is different fromthe (2S,3R)-isomer (B) only in the ester residue R¹, crystallizing the(2R,3S)-isomer (A) from the solution up to the extent that the(2R,3S)-isomer (A) is crystallized without the precipitation of the(2S,3R)-isomer (B) due to the presence of the ester compound (B′) thoughthe (2S,3R)-isomer (B) would precipitate if the ester compound (B′) werenot present, and isolating the crystals of the (2R,3S)-isomer (A).
 2. Aprocess for preparing a (2S,3S)-isomer of a 1,5-benzothiazepinederivative of the formula (II):

wherein ring A and ring B are independently a substituted orunsubstituted benzene ring, and R² is a 2-(dimethylamino)ethyl group ora group of the formula:

or a pharmaceutically acceptable salt thereof, which comprises (1)obtaining the (2R,3S)-isomer of a trans-3-substituted glycidic acidester compound of the formula (I):

wherein ring A is a substituted or unsubstituted benzene ring, and R¹ isan ester residue by preparing a solution of the (2R,3S)-isomer (A) andthe (2S,3R)-isomer (B) of the ester compound (I) and an ester compound(B′) which is different from the (2S,3R)-isomer (B) only in the esterresidue R¹, crystallizing the (2R,3S)-isomer (A) from the solution up tothe extent that the (2R,3S)-isomer (A) is crystallized without theprecipitation of the (2S,3R)-isomer (B) due to the presence of the estercompound (B′) though the (2S,3R)-isomer (B) would precipitate if theester compound (B′) were not present, and isolating the crystals of the(2R,3S)-isomer (A), and (2) converting the (2R,3S)-isomer (A) to the(2S,3S)-isomer of the 1,5-benzothiazepine derivative or apharmaceutically acceptable salt thereof.
 3. The process of claim 1 or2, wherein the solution further contains a small amount of an estercompound (A′) which is different from the (2R,3S)-isomer (A) only in theester residue R¹ and has the same ester residue as the ester compound.4. The process of claim 3, wherein the ester residue of the(2S,3R)-isomer (B) is a methyl or an ethyl group, and the ester residueof the ester compound (B′) is a member selected from the groupconsisting of: (a) a linear or branched alkyl group which has morecarbon atoms that that of the ester residue R¹ of the (2S,3R)-isomer (B)and which may be substituted by a halogen atom; (b) an alkoxyalkyl groupwhich may be substituted by a halogen atom, and (c) an arylalkyl groupwhich may be substituted by a linear or branched lower alkyl group, alinear or branched lower alkoxy group or a halogen atom.
 5. The processof claim 1 or 2, wherein the molar ratio of (2R,3S)-isomer(A)/(2S,3R)-isomer (B) in the solution is from 4/6 to 10/1, and themolar ratio of ester compound (B′)/(2S,3R)-isomer (B) in the solution isfrom 5/3 to 10/1.
 6. The process of claim 3, wherein the molar ratio ofester compound (A′)/(2R/3S)-isomer (A) is at most 9/35 of that of estercompound (B′)/(2S/3R)-isomer (B).
 7. The process of claim 1 or 2,wherein the solvent of the solution is a member selected from the groupconsisting of an alcohol solvent, an ether solvent, an aromatichydrocarbon solvent which may be substituted by a halogen atom, analiphatic hydrocarbon solvent which may be substituted by a halogen atomand an ester solvent.
 8. The process of claim 1 or 2, wherein theconcentration of (2R,3S)-isomer (A) in the solution prior tocrystallizing is from 0.5 to 4 moles/liter, and the crystallization iscarried out at a temperature of −30 to +15° C.
 9. The process of claim 1or 2, wherein the solution to be subjected to the crystallization is asolution obtained by transesterifying the (2S,3R)-isomer (B) in thesolution of the (2R,3S)-isomer (A) and the (2S,3R)-isomer (B) to anester compound (B′) in the presence of an enzyme having astereoselective transesterification ability by the use of an alcohol.10. The process of claim 9, wherein the enzyme has an E-value of atleast 20 measured when the conversion rate of transesterificationreaction is 10%.
 11. The process of claim 9, wherein thetransesterification is conducted so that the molar ratio of estercompound (B′)/(2S,3R)-isomer (B) is from 5/3 to 10/1.
 12. The process ofclaim 1 or 2, wherein ring A is a 4-methoxyphenyl group, the esterresidue of the (2S,3R)-isomer (B) is a methyl group, and the esterresidue of the ester compound (B′) is a n-butyl group.
 13. The processof claim 12, wherein ring B is an unsubstituted benzene ring, and R² isa 2-(dimethylamino)ethyl group.
 14. In a process for preparing a(2S,3S)-1,5-benzothiazepine derivative of the formula (II):

wherein ring A and ring B are independently a substituted orunsubstituted benzene ring, and R² is a 2- (dimethylamino) ethyl groupor a group of the formula:

or a pharmaceutically acceptable salt thereof, the improvementcomprising using as the starting material a (2R,3S)-isomer of atrans-3-substituted glycidic acid ester compound of the formula (I):

wherein ring A is a substituted or unsubstituted benzene ring, and R¹ isan ester residue, which is obtained by subjecting a mixture of(2R,3S)-isomer (A) and (2S,3R)-isomer (B) of the ester compound (I),both of which are optical isomers due to the asymmetric carbons at the2- and 3-positions, to transesterification in the presence of an alcoholand an enzyme having a stereoselective transesterification ability,thereby transesterifying the (2S,3R)-isomer (B) with the alcohol toproduce an ester compound (B′) which is different from the(2S,3R)-isomer (B) only in the ester residue R¹ until the molar ratio ofester compound (B′)/(2S,3R)-isomer (B) falls within the range of 13/7 to7.8/1, crystallizing the (2R,3S)-isomer (A) from the resulting solutioncontaining the (2R,3S)-isomer (A), the untransesterified (2S,3R)-isomer(B) and the ester compound (B′), and isolating the (2R,3S)-isomer (A)having an optical purity of at least 99% in a yield of at least 75%based on the initial amount of (2R,3S)-isomer (A).
 15. A process forpreparing a (2S,3S)-isomer of a 1,5-benzothiazepine derivative of theformula (II):

wherein ring A and ring B are independently a substituted orunsubstituted benzene ring, and R is a 2-(dimethylamino)ethyl group or agroup of the formula:

or a pharmaceutically acceptable salt thereof, which comprises (1)obtaining the (2R,3S)-isomer of a trans-3-substituted glycidic acidester compound of the formula (I):

wherein ring A is a substituted or unsubstituted benzene ring, and R¹ isan ester residue by subjecting a mixture of (2R,3S)-isomer (A) and(2S,3R)-isomer (B) of the ester compound (I) to transesterification inthe presence of an alcohol and an enzyme having a stereoselectivetransesterification ability, thereby transesterifying the (2S,3R)-isomer(B) with the alcohol to produce an ester compound (B′) which isdifferent from the (2S,3R)-isomer (B) only in the ester residue R¹ untilthe molar ratio of ester compound (B′)/(2S,3R)-isomer (B) falls withinthe range of 13/7 to 7.8/1, crystallizing the (2R,3S)-isomer (A) fromthe resulting solution containing the isomer (A), the unesterified(2S,3R)-isomer (B) and the ester compound (B′), and isolating the isomer(A) having an optical purity of at least 99% in a yield of at least 75%based on the initial amount of (2R,3S)-isomer (A), and (2) convertingthe (2R,3S)-isomer (A) to the (2S,3S)-isomer of the 1,5-benzothiazepinederivative or a pharmaceutically acceptable salt thereof.
 16. Theprocess of claim 14 or 15, wherein the enzyme has an E-value of at least20 measured when the conversion rate of transesterification reaction is10%.
 17. The process of claim 14 or 15, wherein the enzyme is anesterase derived from Serratia marcescens, the molar ratio of estercompound (B′)/(2S,3R)-isomer (B) is from 2/1 to 8/2, and the yield ofthe (2R,3S)-isomer (A) is at least 80%.
 18. The process of claim 14 or15, wherein ring A is a 4-methoxyphenyl group, the ester residue of theisomer (B) is a methyl group, and the ester residue of the estercompound (B′) is a n-butyl group.
 19. The process of claim 18, whereinring B is an unsubstituted benzene ring, and R² is a2-(dimethylamino)ethyl group.
 20. In a process for preparing a(2R,3R)-1,5-benzothiazepine derivative of the formula (III):

wherein ring A and ring B are independently a substituted orunsubstituted benzene ring, or a pharmaceutically acceptable saltthereof, the improvement comprising using as the starting material a(2S,3R)-isomer of a trans-3-substituted glycidic acid ester compound ofthe formula (I):

wherein ring A is a substituted or unsubstituted benzene ring, and R¹ isan ester residue, which is obtained by preparing a solution of(2S,3R)-isomer (A) and (2R,3S)-isomer (B) of the ester compound (I),both of which are optical isomers due to the asymmetric carbons at the2- and 3-positions, and an ester compound (B′) which is different fromthe (2R,3S)-isomer (B) only in the ester residue R1, crystallizing the(2S,3R)-isomer (A) from the solution up to the extent that the(2S,3R)-isomer (A) is crystallized without the precipitation of the(2R,3S)-isomer (B) due to the presence of the ester compound (B′) thoughthe (2R,3S)-isomer (B) would precipitate if the ester compound (B′) werenot present, and isolating the crystals of the (2S,3R)-isomer (A).
 21. Aprocess for preparing a (2R,3R)-isomer of 1,5-benzothiazepine derivativeof the formula (III):

wherein ring A and ring B are independently a substituted orunsubstituted benzene ring, or a pharmaceutically acceptable saltthereof, which comprises (1) obtaining a (2S,3R)-isomer of atrans-3-substituted glycidic acid ester compound of the formula (I):

wherein ring A is a substituted or unsubstituted benzene ring, and R¹ isan ester residue by preparing a solution of (2S,3R)-isomer (A) and(2R,3S)-isomer (B) of the ester compound (I) and an ester compound (B′)which is different from the (2R,3S)-isomer (B) only in the ester residueR¹, crystallizing the (2S,3R)-isomer (A) from the solution up to theextent that the (2S,3R)-isomer (A) is crystallized without theprecipitation of the (2R,3S)-isomer (B) due to the presence of the estercompound (B′) though the (2R,3S)-isomer (B) would precipitate if theester compound (B′) were not present, and isolating the crystals of the(2S,3R)-isomer (A), and (2) converting the (2S,3R)-isomer (A) to the(2R,3R)-isomer of the 1,5-benzothiazepine derivative or apharmaceutically acceptable salt thereof.
 22. The process of claim 20 or21, wherein the solution further contains a small amount of an estercompound (A′) which is different from the (2S,3R)-isomer (A) only in theester residue R¹ and has the same ester residue as the ester compound(B′).
 23. The process of claim 22, wherein the ester residue of the(2R,3S)-isomer (B) is a methyl or an ethyl group, and the ester residueof the ester compound (B′) is a member selected from the groupconsisting of: (a) a linear or branched alkyl group which has morecarbon atoms than that of the ester residue R¹ of the (2R,3S)-isomer (B)and which may be substituted by a halogen atom; (b) an alkoxyalkyl groupwhich may be substituted by a halogen atom, and (c) an arylalkyl groupwhich may be substituted by a linear or branched lower alkyl group, alinear or branched lower alkoxy group or a halogen atom.
 24. The processof claim 20 or 21, wherein the molar ratio of (2S,3R)-isomer(A)/(2R,3S)-isomer (B) in the solution is from 4/6 to 10/1, and themolar ratio of ester compound (B′)/(2R,3S)-isomer (B) in the solution isfrom 5/3 to 10/1.
 25. The process of claim 22, wherein the molar ratioof ester compound (A′)/(2S/3R)-isomer (A) is at most 9/35 of that ofester compound (B′)/(2R,3S)-isomer (B).
 26. The process of claim 20 or21, wherein the solvent of the solution is a member selected from thegroup consisting of an alcohol solvent, an ether solvent, an aromatichydrocarbon solvent which may be substituted by a halogen atom, analiphatic hydrocarbon solvent which may be substituted by a halogen atomand an ester solvent.
 27. The process of claim 20 or 21, wherein theconcentration of (2S,3R)-isomer (A) in the solution prior tocrystallizing is from 0.5 to 4 moles/liter, and the crystallization iscarried out at a temperature of −30 to +15° C.
 28. The process of claim20 or 21, wherein the solution to be subjected to the crystallization isa solution obtained by transesterifying the (2R,3S)-isomer (B) in thesolution of the (2S,3R)-isomer (A) and the (2R,3S)-isomer (B) to anester compound (B′) in the presence of an enzyme having astereoselective transesterification ability by the use of an alcohol.29. The process of claim 28, wherein the enzyme has an E-value of atleast 20 measured when the conversion rate of transesterificationreaction is 10%.
 30. The process of claim 28, wherein thetransesterification is conducted so that the molar ratio of estercompound (B′)/(2S,3R)-isomer (B) is from 5/3 to 10/1.
 31. The Process ofclaim 20 or 21, wherein ring A is a 4-methoxyphenyl group, ring B is anunsubstituted benzene ring, the ester residue of the (2R,3S)-isomer (B)is a methyl group, and the ester residue of the ester compound (B′) is an-butyl group.
 32. In a process for preparing a(2R,3R)-1,5-benzothiazepine derivative of the formula (III):

wherein ring A and ring B are independently a substituted orunsubstituted benzene ring, or a pharmaceutically acceptable saltthereof, the improvement comprising using as the starting material a(2S,3R)-isomer of a trans-3-substituted glycidic acid ester compound ofthe formula (I):

wherein ring A is a substituted or unsubstituted benzene ring, and R¹ isan ester residue, which is obtained by subjecting a mixture of(2S,3R)-isomer (A) and (2R,3S)-isomer (B) of the ester compound (I),both of which are optical isomers due to the asymmetric carbons at the2- and 3-positions, to transesterification in the presence of an alcoholand an enzyme having a stereoselective transesterification ability,thereby transesterifying the (2R,3S)-isomer (B) with the alcohol toproduce an ester compound (B′) which is different from the(2R,3S)-isomer (B) only in the ester residue R¹ until the molar ratio ofester compound (B′)/(2R,3S)-isomer (B) falls within the range of 13/7 to7.8/1, crystallizing the (2S,3R)-isomer (A), from the resulting solutioncontaining the (2S,3R)-isomer (A), the untransesterified (2R,3S)-isomer(B) and the ester compound (B′), and isolating the (2S,3R)-isomer (A)having an optical purity of at least 99% in a yield of at least 75%based on the initial amount of (2S,3R)-isomer (A).
 33. A process forpreparing a (2R,3R)-isomer of a 1,5-benzothiazepine derivative of theformula (III):

wherein ring A and ring B are independently a substituted orunsubstituted benzene ring, or a pharmaceutically acceptable saltthereof, which comprises (1) obtaining the (2S,3R)-isomer of atrans-3-substituted glycidic acid ester compound of the formula (I):

wherein ring A is a substituted or unsubstituted benzene ring, and R¹ isan ester residue by subjecting a mixture of (2S,3R)-isomer (A) and(2R,3S)-isomer (B) of the ester compound (I) to transesterification inthe presence of an alcohol and an enzyme having a stereoselectivetransesterification ability, thereby transesterifying the (2R,3S)-isomer(B) with the alcohol to produce an ester compound (B′) which isdifferent from the (2R,3S)-isomer (B) only in the ester residue R¹ untilthe molar ratio of ester compound (B′)/(2R,3S)-isomer (B) falls withinthe range of 13/7 to 7.8/1, crystallizing the (2S,3R)-isomer (A) fromthe resulting solution containing the isomer (A), the unesterified(2R,3S)-isomer (B) and the ester compound (B′), and isolating the isomer(A) having an optical purity of at least 99% in a yield of at least 75%based on the initial amount of (2S,3R)-isomer (A), and (2) convertingthe (2S,3R)-isomer (A) to the (2S,3R)-isomer of the 1,5-benzothiazepinederivative or a pharmaceutically acceptable salt thereof.
 34. Theprocess of claim 32 or 33, wherein the enzyme has an E-value of at least20 measured when the conversion rate of transesterification reaction is10%.
 35. The process claim 32 or 33, wherein ring A is a 4-methoxyphenylgroup, ring B is an unsubstituted benzene ring, the ester residue of theisomer (B) is a methyl group, and the ester residue of the estercompound (B′) is a n-butyl group.